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Sample records for diphtheria toxoid

  1. Improved specificity of a multiplex immunoassay for quantitation of anti-diphtheria toxin antibodies with the use of diphtheria toxoid.

    PubMed

    van Gageldonk, Pieter G M; von Hunolstein, Christina; van der Klis, Fiona R M; Berbers, Guy A M

    2011-07-01

    A nonspecific binding of antibodies to diphtheria toxin, especially in adult serum samples, was observed in our diphtheria-tetanus-pertussis multiplex immunoassay (DTaP4 MIA). This can be significantly reduced by the use of diphtheria toxoid, achieving a good correlation with the Vero cell neutralization test and the toxin binding inhibition assay.

  2. Comparison of gel filtration and ammonium sulphate precipitation in the purification of diphtheria toxin and toxoid.

    PubMed

    Møyner, K; Christiansen, G

    1984-02-01

    Crude diphtheria toxin and toxoid were subjected to purification by gel filtration and stepwise ammonium sulphate precipitation. The various fractions obtained by the purification procedures were studied by immunological methods. A high molecular weight fraction of glycoprotein nature was present in all of the crude preparations studied. The fraction was antigenically non-identical with the real toxin or toxoid and did not have its origin in the culture medium. It showed a long flocculation time when tested against equine diphtheria toxoid antiserum. The fraction could be removed from the crude preparations by gel filtration or by precipitation with 21% (w/v) ammonium sulphate. When comparing toxoids purified by each of these methods, the method of gel filtration resulted in a somewhat higher degree of purity, suggesting that this method would be more suitable than the AS precipitation method for the purification of diphtheria toxoid.

  3. Quantitative estimation of diphtheria and tetanus toxoids. 5. Comparative assays in mice and in guinea-pigs of two diphtheria toxoid preparations.

    PubMed

    Lyng, J; Heron, I

    1991-10-01

    Two freeze-dried international reference diphtheria toxoids of different origin were compared in biological assays in guinea-pigs and mice under different adjuvant conditions. When the antigenic content in the two toxoids was used as denominator for determination of relative potency, that is to say quantitation of immunogenic power per unit amount of antigen, the design of the animal assay proved to have a major influence. Similar observations have been made previously also for tetanus vaccines. It is concluded that diphtheria vaccines as well as tetanus vaccines can hardly be quantitated unambiguously using the currently recommended potency assays in animals. A new scheme for control of toxoid vaccine production is suggested, with more emphasis on the control of the bulk purified toxoid, which would make the release of final products more simple and rapid.

  4. International collaborative studies on potency assays of diphtheria and tetanus toxoids.

    PubMed

    Van Ramshorst, J D; Sundaresan, T K; Outschoorn, A S

    1972-01-01

    Collaborative studies showed that relative potency assays for a particular type of diphtheria toxoid (adsorbed) and for tetanus toxoid (plain and adsorbed) gave very similar results, whether the assays were carried out by toxin challenge or by antitoxin titration after immunization of experimental animals with graded doses of toxoid. The same numerical results were obtained with a scoring system as with a system based on survivals only. Although skin tests were used on a very limited scale in these studies, it seems likely that they could replace lethal tests for the diphtheria challenge assays.For both tetanus and diphtheria, the adsorbed toxoid gave a higher relative potency when combined with other antigens than as a single toxoid. Both mice and guinea-pigs were used for the lethal challenge test of adsorbed tetanus toxoid. For the single tetanus toxoid the results were the same, but for the combined toxoid (DPT vaccine) the mouse assay results were about twice those of guinea-pig assays.

  5. Comparison of CRM197, diphtheria toxoid and tetanus toxoid as protein carriers for meningococcal glycoconjugate vaccines.

    PubMed

    Tontini, M; Berti, F; Romano, M R; Proietti, D; Zambonelli, C; Bottomley, M J; De Gregorio, E; Del Giudice, G; Rappuoli, R; Costantino, P; Brogioni, G; Balocchi, C; Biancucci, M; Malito, E

    2013-10-01

    Glycoconjugate vaccines are among the most effective and safest vaccines ever developed. Diphtheria toxoid (DT), tetanus toxoid (TT) and CRM197 have been mostly used as protein carriers in licensed vaccines. We evaluated the immunogenicity of serogroup A, C, W-135 and Y meningococcal oligosaccharides conjugated to CRM197, DT and TT in naïve mice. The three carriers were equally efficient in inducing an immune response against the carbohydrate moiety in immunologically naïve mice. The effect of previous exposure to different dosages of the carrier protein on the anti-carbohydrate response was studied using serogroup A meningococcal (MenA) saccharide conjugates as a model. CRM197 showed a strong propensity to positively prime the anti-carbohydrate response elicited by its conjugates or those with the antigenically related carrier DT. Conversely in any of the tested conditions TT priming did not result in enhancement of the anti-carbohydrate response elicited by the corresponding conjugates. Repeated exposure of mice to TT or to CRM197 before immunization with the respective MenA conjugates resulted in a drastic suppression of the anti-carbohydrate response in the case of TT conjugate and only in a slight reduction in the case of CRM197. The effect of carrier priming on the anti-MenA response of DT-based conjugates varied depending on their carbohydrate to protein ratio. These data may have implications for human vaccination since conjugate vaccines are widely used in individuals previously immunized with DT and TT carrier proteins.

  6. Quantitative estimation of diphtheria and tetanus toxoids. 6. Use of different antibody titration methods for evaluation of immunogenicity in animals during potency assay of diphtheria toxoid.

    PubMed

    Lyng, J; Heron, I

    1992-06-01

    Two diphtheria toxoid preparations were compared in potency assays in guinea-pigs using different methods for evaluation of the responses to vaccination. The methods used were the direct skin challenge (Schick test) and ELISA and VERO cell titration of antibodies. The different evaluation methods resulted in the same relative potencies between the toxoids. It was observed that when first-vaccination sera were compared with a second-vaccination serum, the relative antibody concentration depended on whether ELISA or VERO cell titration was used.

  7. Short-term booster effect of diphtheria toxoid in initially long-term protected individuals.

    PubMed

    Danilova, Elena; Shiryayev, Alexey; Skogen, Vegard; Kristoffersen, Einar Klaeboe; Sjursen, Haakon

    2005-02-10

    The main objective of this study was to investigate the booster antibody response in individuals with initially high levels of diphtheria antitoxin. Sixty individuals eligible for the routine booster by the age of 18 years each received a single dose of 5 Lf of diphtheria toxoid in diphtheria-tetanus vaccine. A double antigen ELISA was used for the assessment of the antibody levels. Chaotropic disruption in paired ELISA was used to test antibody avidity. The ratio between initial and maximum antibody concentrations after 1 month was >10 times higher and after 6 months still four times higher in those with initial antibody levels <1 IU/ml. In individuals with initial antibody levels >/=1 IU/ml a two-fold decrease was observed after 6 months compared to the initial levels. Thus, vaccination of individuals with initial long-term protection against diphtheria (antibody levels >/=1 IU/ml) is unnecessary and should be avoided.

  8. Calibration of replacement international standards of diphtheria and tetanus toxoids for use in flocculation test.

    PubMed

    Preneta-Blanc, R; Rigsby, P; Wilhelmsen, E Sloth; Tierney, R; Brierley, M; Sesardic, D

    2008-09-01

    The 1st International Reference Reagents (IRR) of Diphtheria and Tetanus Toxoids for Flocculation Test (DIFT and TEFT) were established by the WHO in 1988. These reagents are essential for the standardization of assays used to calculate Lf units of toxoids. Candidate replacement materials were provided by several European vaccine manufacturers and were formulated and freeze-dried at NIBSC. This paper provides a summary of the results of an international collaborative study including 18 laboratories from 16 countries, which examined the candidate replacement materials in a variety of methods. Materials 02/176 and 04/150 were proposed and adopted by the Expert Committee on Biological Standardization of WHO in October 2007 as 2nd WHO International Standards of Diphtheria and Tetanus Toxoid for use in Flocculation Test. The replacement standards were assigned the value of 1100 and 690Lf/ampoule, respectively, based on results of flocculation tests carried out using provided reagents. Material coded 02/176 fully complied with the WHO specifications for stability, residual moisture content, precision of fill and sterility. Stability of material coded 04/150 was slightly lower than expected but predictions were based only on 2-year data and were to be further monitored, post-adoption.

  9. Physico-chemical properties of Salmonella typhi Vi polysaccharide-diphtheria toxoid conjugate vaccines affect immunogenicity.

    PubMed

    An, So Jung; Yoon, Yeon Kyung; Kothari, Sudeep; Kothari, Neha; Kim, Jeong Ah; Lee, Eugene; Kim, Deok Ryun; Park, Tai Hyun; Smith, Greg W; Carbis, Rodney

    2011-10-13

    In this study it was demonstrated that the immunogenicity of Vi polysaccharide-diphtheria toxoid conjugates was related to the physical and chemical structure of the conjugate. Conjugates were prepared in two steps, firstly binding adipic acid dihydrazide (ADH) spacer molecules to diphtheria toxoid (DT) carrier protein then secondly binding varying amounts of this derivatized DT to a fixed amount of Vi capsular polysaccharide purified from Salmonella enterica Serovar Typhi. As the amount of DT bound to the Vi increased the size of the conjugate increased but also the degree of cross-linking increased. The immunogenicity of the conjugates was tested in mice and measured by ELISA for anti Vi and anti DT IgG responses, and the results revealed a trend that as the amount of DT bound to the Vi increased the anti Vi responses increased. This study establishes a correlation between physico-chemical characteristics of the conjugate and the magnitude of the anti Vi and anti DT responses.

  10. Comparative quantitation for the protein content of diphtheria and tetanus toxoids by DC protein assay and Kjeldahl method.

    PubMed

    Doshi, J B; Ravetkar, S D; Ghole, V S; Rehani, K

    2003-09-01

    DPT, a combination vaccine against diphtheria, tetanus and pertussis is available since many years and still continued in the national immunisation schedule of many countries. Although highly potent, reactions to DPT vaccine are well known, mainly attributed to the factors like Pertussis component, aluminum adjuvant and lower purity of tetanus and diphtheria toxoids. The latter most important aspect has become a matter of concern, specially for the preparation of next generation combination vaccines with more number of antigens in combination with DPT. Purity of toxoid is expressed as Lf (Limes flocculation) per mg of protein nitrogen. The Kjeldahl method (KM) of protein nitrogen estimation suggested by WHO and British Pharmacopoeia is time consuming and less specific. Need has been felt to explore an alternative method which is quick and more specific for toxoid protein determination. DC (detergent compatible) protein assay, an improved Lowry's method, has been found to be much more advantageous than Kjeldahl method.

  11. Potential protective immunogenicity of tetanus toxoid, diphtheria toxoid and Cross Reacting Material 197 (CRM197) when used as carrier proteins in glycoconjugates.

    PubMed

    Bröker, Michael

    2016-03-03

    When tetanus toxoid (TT), diphtheria toxoid (DT) or Cross Reacting Material 197 (CRM197), a non-toxic diphtheria toxin mutant protein, are used as carrier proteins in glycoconjugate vaccines, these carriers induce a protein specific antibody response as measured by in vitro assays. Here, it was evaluated whether or not glycoconjugates based on TT, DT or CRM197 can induce a protective immune response as measured by potency tests according to the European Pharmacopoeia. It could be shown, that the conjugate carriers TT and DT can induce a protective immune response against a lethal challenge by toxins in animals, while glycoconjugates based on CRM197 failed to induce a protective immune response. Opportunities for new applications of glycoconjugates are discussed.

  12. Quantitative estimation of diphtheria and tetanus toxoids. 2. Single radial immuno-diffusion tests (Mancini) and rocket immuno-electrophoresis test in comparison with the flocculation test.

    PubMed

    Ljungqvist, L; Lyng, J

    1987-01-01

    The concentration in Lf units, of an unknown diphtheria or tetanus toxoid preparation is estimated in the flocculation test relative to reference preparations of tetanus and diphtheria antitoxins, respectively. By replacing the antitoxin reference preparations with toxoid reference preparations it should be possible to use immunological methods other than the flocculation test for the quantitative estimation of toxoids in Lf units. A number of diphtheria and tetanus toxoids were tested by rocket immuno-electrophoresis and single radial immuno-diffusion (Mancini test). The concentrations of the unknown toxoids were expressed relative to a diphtheria toxoid calibrated in Lf units (DIFT) and a tetanus toxoid calibrated in Lf units (TEFT), respectively. These two toxoid preparations are regarded as candidates for establishment as international standard preparations. The results obtained in the two tests were compared with those obtained in the flocculation test. In most cases the differences between the results did not exceed 10%. It is concluded, therefore, that the rocket electrophoresis or the radial immuno-diffusion tests can be used as alternatives to the flocculation test.

  13. Technical and diagnostic performance of five commercial anti-diphtheria toxoid IgG enzyme-linked immunosorbent assay kits.

    PubMed

    Faruq, A; Dadson, L; Cox, H; Alcock, F; Parker, A R

    2010-10-01

    The technical and diagnostic performances of five commercially available enzyme-linked immunosorbent assays for the measurement of anti-diphtheria toxoid IgG antibodies were evaluated. There was good agreement between the relative sensitivities of the five assays, but the relative specificity of one of the assays differed from that of the other four assays. Three of the five assays possessed recoveries of the international reference material NIBSC 00/496 within the range of 90% to 110% at antibody levels >0.1 IU/ml. The data suggest that there are manufacture-dependent differences in relative sensitivity, specificity, and accuracy for the determination of anti-diphtheria toxoid IgG antibodies that could result in different diagnostic interpretations.

  14. A Bordetella pertussis proteoliposome induces protection in mice without affecting the immunogenicity of diphtheria and tetanus toxoids in a trivalent formulation.

    PubMed

    Castillo, Sonsire Fernández; Chovel, Mario Landys; Hernández, Niurka Gutiérrez; González, Lorena Corcho; Blanco, Amaya; Hernández, Daily Serrano; Medina, Mildrey Fariñas; Tito, Maydelis Álvarez; Quiñoy, José Luis Pérez

    2016-07-01

    In this study, a formulation of Bordetella pertussis proteoliposome (PLBp), diphtheria, and tetanus toxoids and alum (DT-PLBp) was evaluated as a trivalent vaccine candidate in BALB/c mice. Vaccine-induced protection was estimated using the intranasal challenge for pertussis and enzyme-linked immunosorbent assay fvto assess serological responses for diphtheria or tetanus. Both, diphtheria-tetanus-whole cell pertussis (DTP) and diphtheria-tetanus vaccines (DT) were used as controls. Animals immunized with DT-PLBp, PLBp alone, and DTP showed total reduction of CFU in lungs 7 days after intranasal challenge. Likewise, formulations DT-PLBp, DTP, and DT elicited antibody levels ≥2 IU/mL against tetanus and diphtheria, considered protective when neutralization tests are used. Overall, results showed that combination of PLBp with tetanus and diphtheria toxoids did not affect the immunogenicity of each antigen alone.

  15. Evaluation of a single dose of diphtheria toxoid among adults in the Republic of Georgia, 1995: immunogenicity and adverse reactions.

    PubMed

    Khetsuriani, N; Music, S; Deforest, A; Sutter, R W

    2000-02-01

    To determine the immunogenicity and safety of a single dose of diphtheria toxoid among adults, blood samples for detecting serum antitoxin levels were obtained from 18- to 59-year-old subjects (n=248) before and 30 days after immunization with Td (tetanus-diphtheria toxoids; manufactured by Serum Institute of India). By day 30, the seroprevalence of antitoxin levels >/=0.1 IU/mL increased from 22.6% to 81.5%; median antitoxin levels increased from 0.01 to 4.0 IU/mL. These parameters were lowest among subjects who were 40-59 years old, especially among those 40-49 years old. Adverse reactions (local redness, swelling, induration, fever>39 degrees C) were reported by 5.3% of participants. Our findings suggest that, in general, one dose of the Indian-produced Td vaccine is efficacious and safe in inducing an adequate immune response against diphtheria in adults; however, in Georgia, persons 40-59 years old, especially those 40-49 years old, will require additional doses of toxoid to achieve protective levels of antitoxin.

  16. Evidence of increased carriage of Corynebacterium spp. in healthy individuals with low antibody titres against diphtheria toxoid.

    PubMed

    Bergamini, M; Fabrizi, P; Pagani, S; Grilli, A; Severini, R; Contini, C

    2000-08-01

    This study evaluated whether a correlation exists between carriage of corynebacteria and the lack of immunity to diphtheria toxoid. Samples of both nasal and pharyngeal secretions were taken from 500 apparently healthy subjects of both sexes and of all ages and inoculated onto Tinsdale's medium. A serum sample was also taken for ELISA test to determine the titre of diphtheria toxin antibodies. None of the subjects carried Corynebacterium diphtheriae. Ninety-three strains of Corynebacterium spp. were isolated from 93 subjects and 86 of these were classified to species or group level by biochemical tests. C. xerosis was the most common (25.8%) followed by C. pseudodiphthericum (16.1%), C. jeikeium and C. striatum (both 10.8%), and C. urealyticum (9.7%). Three other species accounted for approximately 20% of strains and seven were unclassified as biochemically atypical corynebacteria. Non-protective antibodies to diphtheria toxin were found in 80 of the 93 subjects and a strong statistical association was demonstrated between carriage of corynebacteria and non-protective levels of anti-toxin antibodies. The remaining 13 subjects had protective levels of antitoxin antibodies. In contrast, only 45 of the 407 non-colonized subjects had non-protective antitoxin titres. The prevalence of carriage increased with age among males as did the percentage of non-protected subjects. The prevalence of female carriers of corynebacteria was significantly lower. Serum samples from 12 subjects with different antibody titres to diphtheria toxoid reacted to varying degrees with whole-cell lysates of a number of species of corynebacteria. The results suggest that a causal relationship may exist between nasopharyngeal carriage of corynebacteria and a low anti-diphtheria toxin immune response.

  17. Diphtheria toxoid loaded poly-(epsilon-caprolactone) nanoparticles as mucosal vaccine delivery systems.

    PubMed

    Singh, Jasvinder; Pandit, Sreenivas; Bramwell, Vincent W; Alpar, H Oya

    2006-02-01

    Poly-(epsilon-caprolactone) (PCL), a poly(lactide-co-glycolide) (PLGA)-PCL blend and co-polymer nanoparticles encapsulating diphtheria toxoid (DT) were investigated for their potential as a mucosal vaccine delivery system. The nanoparticles, prepared using a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation method, demonstrated release profiles which were dependent on the properties of the polymers. An in vitro experiment using Caco-2 cells showed significantly higher uptake of PCL nanoparticles in comparison to polymeric PLGA, the PLGA-PCL blend and co-polymer nanoparticles. The highest uptake mediated by the most hydrophobic nanoparticles using Caco-2 cells was mirrored in the in vivo studies following nasal administration. PCL nanoparticles induced DT serum specific IgG antibody responses significantly higher than PLGA. A significant positive correlation between hydrophobicity of the nanoparticles and the immune response was observed following intramuscular administration. The positive correlation between hydrophobicity of the nanoparticles and serum DT specific IgG antibody response was also observed after intranasal administration of the nanoparticles. The cytokine assays showed that the serum IgG antibody response induced is different according to the route of administration, indicated by the differential levels of IL-6 and IFN-gamma. The nanoparticles eliciting the highest IgG antibody response did not necessarily elicit the highest levels of the cytokines IL-6 and IFN-gamma.

  18. Post-licensure safety surveillance study of routine use of tetanus toxoid, reduced diphtheria toxoid and 5-component acellular pertussis vaccine

    PubMed Central

    Baxter, Roger; Hansen, John; Timbol, Julius; Pool, Vitali; Greenberg, David P.; Johnson, David R.; Decker, Michael D.

    2016-01-01

    ABSTRACT An observational post-licensure (Phase IV) retrospective large-database safety study was conducted at Kaiser Permanente, a US integrated medical care organization, to assess the safety of Tetanus Toxoid, Reduced Diphtheria Toxoid and 5-Component Acellular Pertussis Vaccine (Tdap5) administered as part of routine healthcare among adolescents and adults. We evaluated incidence rates of various clinical events resulting in outpatient clinic, emergency department (ED), and hospital visits during various time intervals (windows) following Tdap5 vaccination using 2 pharmacoepidemiological methods (risk interval and historic cohort) and several screening thresholds. Plausible outcomes of interest with elevated incidence rate ratios (IRRs) were further evaluated by reviewing individual patient records to confirm the diagnosis, timing (temporal relationship), alternative etiology, and other health record details to discern possible relatedness of the health events to vaccination. Overall, 124,139 people received Tdap5 vaccine from September 2005 through mid-October 2006, and 203,154 in the comparison cohort received a tetanus and diphtheria toxoid adsorbed vaccine (and no live virus vaccine) during the year prior to initiation of this study. In the outpatient, ED and hospital databases, respectively, we identified 11/26, 179/700 and 187/700 unique health outcomes with IRRs significantly >1.0. Among the same unique health outcomes in the outpatient, ED, and hospital databases, 9, 146, and 385, respectively, had IRRs significantly <1.0. Further scrutiny of the outcomes with elevated IRRs did not reveal unexpected signals of adverse outcomes related to vaccination. In conclusion, Tdap5 vaccine was found to be safe among this large population of adolescents and adults. PMID:27388557

  19. Transcutaneous immunization with cross-reacting material CRM(197) of diphtheria toxin boosts functional antibody levels in mice primed parenterally with adsorbed diphtheria toxoid vaccine.

    PubMed

    Stickings, Paul; Peyre, Marisa; Coombes, Laura; Muller, Sylviane; Rappuoli, Rino; Del Giudice, Giuseppe; Partidos, Charalambos D; Sesardic, Dorothea

    2008-04-01

    Transcutaneous immunization (TCI) capitalizes on the accessibility and immunocompetence of the skin, elicits protective immunity, simplifies vaccine delivery, and may be particularly advantageous when frequent boosting is required. In this study we examined the potential of TCI to boost preexisting immune responses to diphtheria in mice. The cross-reacting material (CRM(197)) of diphtheria toxin was used as the boosting antigen and was administered alone or together with either one of two commonly used mucosal adjuvants, cholera toxin (CT) and a partially detoxified mutant of heat-labile enterotoxin of Escherichia coli (LTR72). We report that TCI with CRM(197) significantly boosted preexisting immune responses elicited after parenteral priming with aluminum hydroxide-adsorbed diphtheria toxoid (DTxd) vaccine. In the presence of LTR72 as an adjuvant, toxin-neutralizing antibody titers were significantly higher than those elicited by CRM(197) alone and were comparable to the functional antibody levels induced after parenteral booster immunization with the adsorbed DTxd vaccine. Time course study showed that high levels of toxin-neutralizing antibodies persisted for at least 14 weeks after the transcutaneous boost. In addition, TCI resulted in a vigorous antigen-specific proliferative response in all groups of mice boosted with the CRM(197) protein. These findings highlight the promising prospect of using booster administrations of CRM(197) via the transcutaneous route to establish good herd immunity against diphtheria.

  20. Use of diphtheria toxoid-tetanus toxoid-acellular pertussis vaccine as a five-dose series. Supplemental recommendations of the Advisory Committee on Immunization Practices (ACIP).

    PubMed

    2000-11-17

    Four vaccines containing diphtheria and tetanus toxoids and acellular pertussis vaccine (DTaP) are currently licensed in the United States for use among infants and young children. As of October 2000, two products, ACEL-IMUNE (a product of Lederle Laboratories) and Tripedia (Aventis Pasteur, Inc.) were licensed for the five-dose DTaP vaccination series. Two other vaccines, Infanrix (SmithKline Beecham Biologicals) and Certiva (North American Vaccine, Inc.) are licensed for the first four doses of the vaccination series, beginning with the primary series at ages 2, 4, and 6 months, and for completing the DTaP series among children who began the series with diphtheria and tetanus toxoids and whole-cell pertussis vaccine. This report supplements the statement from CDC's Advisory Committee on Immunization Practices regarding use of acellular pertussis vaccines and summarizes data regarding reactogenicity of acellular pertussis vaccines when administered as the fourth and fifth consecutive doses. Increases in the frequency and magnitude of local reactions at the injection site with increasing dose number have occurred for all currently licensed DTaP vaccines. Extensive swelling of the injected limb, sometimes involving the entire thigh or upper arm, after receipt of the fourth and fifth doses of DTaP vaccines has been demonstrated for multiple products from different manufacturers. Because data are insufficient regarding the safety, immunogenicity, and efficacy of using DTaP vaccines from different manufacturers in a mixed sequence, ACIP continues to recommend that, whenever feasible, the same brand of DTaP vaccine be used for all doses in the vaccination series. When the vaccine provider does not know or does not have available the type of DTaP vaccine previously administered, any of the licensed DTaP vaccines can be used to complete the vaccine series.

  1. Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines-part 1.

    PubMed

    Winsnes, R; Sesardic, D; Daas, A; Behr-Gross, M-E

    2004-01-01

    A collaborative study on the evaluation of an alternative functional assay, the Vero cell method, to the Ph. Eur. in vivo challenge procedures for potency determination of diphtheria toxoid in 6 different combined vaccines was initiated in January 2001. The study was an extension of a previous study for the validation of serological methods for potency testing of tetanus toxoid vaccines for human use. To allow interim evaluation of test results and to monitor study progress, the project was divided into three consecutive phases. The results of Phase I and II studies are presented in this report. Pre-validation (Phase I) study, performed in two laboratories, indicated that comparable diphtheria potency estimates were obtained in the Ph. Eur. direct intradermal challenge assay in guinea pigs, in Vero cell assay and in indirect ELISA for five vaccines of different potencies (range of estimates: ca. 20-200 IU/ml). The correlation coefficients between the challenge assay and the Vero cell assay corresponded to those between the challenge assay and ELISA, confirming that the antibodies play an important role in protection and that predominantly protective/neutralising antibodies are present in guinea pigs, at the time point investigated. It was observed, for Vero cell assays, that about 16-35 (9-28 in Phase II study) fold lower titre of individual serum samples were obtained when using equine, rather than guinea pig reference serum. The study also provided preliminary information that sera from the same guinea pigs may be used for potency determination of both diphtheria and tetanus toxoid components of vaccines. In Phase II, another five laboratories analysed a subset of the vaccines included in Phase I study plus an additional vaccine. Four laboratories performed the lethal challenge assay and one laboratory carried out the intradermal challenge assay. All laboratories also performed the Vero cell assay and both ELISA for diphtheria antitoxin and ELISA for tetanus

  2. Transcutaneous vaccination using a hydrogel patch induces effective immune responses to tetanus and diphtheria toxoid in hairless rat.

    PubMed

    Matsuo, Kazuhiko; Ishii, Yumiko; Quan, Ying-Shu; Kamiyama, Fumio; Mukai, Yohei; Yoshioka, Yasuo; Okada, Naoki; Nakagawa, Shinsaku

    2011-01-05

    Transcutaneous immunization (TCI) targeting the Langerhans cells (LCs) of the epidermal layer is a promising needle-free, easy-to-use, and non-invasive vaccination method. We developed a hydrogel patch formulation to promote the penetration of antigenic proteins into the stratum corneum. Here, we investigated the characteristics of the immune responses induced by this vaccination method and the vaccine efficacy of TCI using a hydrogel patch containing tetanus and diphtheria toxoids. Our TCI system induced toxoid-specific IgG production in an antigen dose-, patch area-, and application period-dependent manner. Moreover, IgG subclass analysis indicated that our TCI predominantly elicited a Th2-type immune response rather than a Th1-type immune response. Importantly, our TCI system induced antigen-specific immune memory based on the booster effect and showed potent efficacy, comparable to that of subcutaneous immunization in toxin-challenge experiments. On the basis of these results, we are now performing translational research to apply TCI for tetanus and diphtheria.

  3. Diphtheria, Tetanus, and Pertussis (DTaP) Vaccine

    MedlinePlus

    Certiva® (as a combination product containing Diphtheria, Tetanus Toxoids, Acellular Pertussis Vaccine) ... Daptacel® (as a combination product containing Diphtheria, Tetanus Toxoids, Acellular Pertussis Vaccine)

  4. ELISA detection of specific functional antibodies in human serum to Escherichia coli, tetanus toxoid, and diphtheria-tetanus toxoids: normal values for IgG, IgA, and IgM.

    PubMed

    Moen, R C; Oemichen, S L; Kiggens, A J; Hong, R

    1986-01-01

    An inexpensive, easily performed enzyme-linked immunosorbent assay (ELISA) was developed to measure specific IgG, IgA, and IgM antibodies to the common antigens Escherichia coli, diphtheria-tetanus toxoid, and tetanus toxoid. Normal values were established. Classical antibody deficiency disease states were confirmed and delineated by these assays. Additionally, several instances were discovered when functional antibody levels were abnormal when the serum immunoglobulin levels were normal. The use of ELISA assays for antibodies to common antigens provides a useful technique to measure and monitor isotype responses of the humoral immune system.

  5. Changes in TNF and IL-6 production after diphtheria toxoid vaccination: drug modulation of the cytokine levels

    PubMed Central

    Bliacher, M. S.; Danilina, A. V.; Kalashnikova, E. A.; Lopatina, T. K.; Fedorova, I. M.

    1996-01-01

    The effect of vaccination with diphtheria toxoid (AD-M) on TNF and IL-6 production has been studied in humans. In the present study it was demonstrated that immunization with AD-M resulted in changes of in vitro TNF and IL-6 production by peripheral blood mononuclear cells. TNF release was suppressed but IL-6 production was stimulated. On the other hand, serum levels of TNF were markedly increased over a period of 3 weeks. It was also demonstrated that the postvaccinal cytokine production disturbances may be corrected by pretreatment with a new synthetic hexapeptid (Imunofan®). It is possible that the imunofan treatment could prevent some postvaccinal complications. PMID:18475748

  6. Tetanus, Diphtheria (Td) Vaccine

    MedlinePlus

    Decavac® (as a combination product containing Diphtheria, Tetanus Toxoids) ... Tenivac® (as a combination product containing Diphtheria, Tetanus Toxoids) ... Why get vaccinated?Tetanus and diphtheria are very serious diseases. They are rare in the United States today, but people who do become ...

  7. IgG and IgG subclass specific antibody responses to diphtheria and tetanus toxoids in newborns and infants given DTP immunization.

    PubMed

    Dengrove, J; Lee, E J; Heiner, D C; St Geme, J W; Leake, R; Baraff, L J; Ward, J I

    1986-08-01

    To evaluate immune responses to diphtheria and tetanus toxoids in infants we used enzyme-linked immunosorbent assays to detect total IgG and specific IgG-1, IgG-2, IgG-3, and IgG-4 antibody. One group of infants received a newborn dose and subsequently received the usual three doses of DTP. A second group of infants received only the routine dosage at 2, 4, and 6 months of age. In sera acquired at birth, 6, and 9 months of age, there were no statistically significant differences between the two vaccine groups in IgG antibody responses to diphtheria or tetanus, or in IgG subclass tetanus-specific antibody responses. In individual children, tetanus-specific subclass responses were similar in pattern to that for total IgG tetanus antibody, i.e. each IgG subclass response appeared to be regulated by similar mechanisms in that child, but the regulation differed between children. In contrast to a prior study of pertussis immunity, maternally acquired antibody did not significantly affect immune responses to diphtheria or tetanus toxoid by 9 months of age. There was no discernible tolerance due to early tetanus or diphtheria immunization or to high levels of maternally acquired antibody.

  8. Generation of polyclonal catalytic antibodies against cocaine using transition state analogs of cocaine conjugated to diphtheria toxoid.

    PubMed

    Basmadjian, G P; Singh, S; Sastrodjojo, B; Smith, B T; Avor, K S; Chang, F; Mills, S L; Seale, T W

    1995-11-01

    Six novel transition state analogs (TSAs) of cocaine (10-14 and 17) and one non-cocaine, p-aminophenylphosphonyl ester of cyclohexanol (19), were synthesized and characterized by 1H- and 13C-NMR and FAB-MS. (1R)-ecgonine methyl ester or cyclohexanol were subjected to phenylphosphonylation in the presence of dicyclohexyl carbodiimde (DCC) and 4-N,N-dimethyl aminopyridine (4-DMAP). TSA-IV (10), however, was synthesized from norcocaine which was protected with dibromoethane to yield 4 before acid hydrolysis, esterification and phenylphosphonylation were carried out. TSA-III (11) TSA-I (12) and (19), using various length spacer arms, were coupled with the immunogenic protein, diphtheria toxoid (DT). The TSAs coupled with DT were used to immunize mice and after appropriate boosts their sera were tested for the presence and titer of anti-TSA polyclonal antibodies using ELISA. Preliminary results show that the mice immunized with these TSAs produced high titers of polyclonal catalytic antibodies, except for (19), with the ability to hydrolyze the substrate 125I-4'-iodococaine in an in vitro assay, even in the presence of noncatalytic anti-TSA antibodies.

  9. Immunogenicity of a combination vaccine containing diphtheria toxoid, tetanus toxoid, three-component acellular pertussis, hepatitis B, inactivated polio virus, and Haemophilus influenzae type b when given concomitantly with 13-valent pneumococcal conjugate vaccine.

    PubMed

    Gimenez-Sanchez, Francisco; Kieninger, Dorothee M; Kueper, Kathrin; Martinon-Torres, Federico; Bernaola, Enrique; Diez-Domingo, Javier; Steul, Kathrin; Juergens, Christine; Gurtman, Alejandra; Giardina, Peter; Liang, John Z; Gruber, William C; Emini, Emilio A; Scott, Daniel A

    2011-08-11

    Two randomized trials of 13-valent pneumococcal conjugate vaccine (PCV13) relative to PCV7 evaluated the immune responses of coadministered antigens comprising Infanrix(®) hexa/Infanrix(®)-IPV+Hib (diphtheria, tetanus, 3-component acellular pertussis, hepatitis B, inactivated poliovirus, and Haemophilus influenzae type b). After the 3-dose infant series, immunogenic noninferiority was demonstrated for all concomitantly administered antigens between the PCV13 and PCV7 groups. All antigens elicited good booster responses after the toddler dose except pertussis toxoid; however, 99.6% subjects achieved pertussis toxoid protective antibody level ≥5EU/mL in both groups. These results support the concomitant administration of PCV13 and Infanrix hexa/Infanrix-IPV+Hib as part of routine immunization schedules.

  10. Updated recommendations for use of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (Tdap) vaccine in adults aged 65 years and older - Advisory Committee on Immunization Practices (ACIP), 2012.

    PubMed

    2012-06-29

    Since 2005, the Advisory Committee on Immunization Practices (ACIP) has recommended a tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (Tdap) vaccine booster dose for all adolescents aged 11 through 18 years (preferred at 11 through 12 years) and for those adults aged 19 through 64 years who have not yet received a dose. In October 2010, despite the lack of an approved Tdap vaccine for adults aged 65 years and older, ACIP recommended that unvaccinated adults aged 65 years and older be vaccinated with Tdap if in close contact with an infant, and that other adults aged 65 years and older may receive Tdap. In July 2011, the Food and Drug Administration (FDA) approved expanding the age indication for Boostrix (GlaxoSmithKline Biologicals, Rixensart, Belgium) to aged 65 years and older. In February 2012, ACIP recommended Tdap for all adults aged 65 years and older. This recommendation supersedes previous Tdap recommendations regarding adults aged 65 years and older.

  11. Investigation in a murine model of possible mechanisms of enhanced local reactions to post-primary diphtheria-tetanus toxoid boosters in recipients of acellular pertussis-diphtheria-tetanus vaccine

    PubMed Central

    Ochiai, Masaki; Horiuchi, Yoshinobu; Yuen, Chun-Ting; Asokanathan, Catpagavalli; Yamamoto, Akihiko; Okada, Kenji; Kataoka, Michiyo; Markey, Kevin; Corbel, Michael; Xing, Dorothy

    2014-01-01

    In recipients primed with acellular pertussis diphtheria-tetanus combined vaccine (DTaP) an increased incidence of severe local reactions with extensive redness/swelling has been reported for each subsequent dose of diphtheria-tetanus based combination vaccine given as a booster. This has been attributed to residual active pertussis toxin (PT) in the primary vaccine. In this study, we investigated the possible contribution of the A-subunit enzymatic activity and the B-oligomer carbohydrate binding activity of residual PT in DTaP to local reactions in a murine model using Japanese DTaP batches produced before and after the introduction of a test for reversion of pertussis toxoid to toxin. Residual PT activity was correlated with the B-oligomer carbohydrate binding activity. The in vivo mouse footpad swelling model assay indicated that the B-oligomer carbohydrate binding activity and possibly other factors were associated with intensified sensitization to local reaction following diphtheria toxoid booster. PMID:25424818

  12. Measurement of antibodies to pneumococcal, meningococcal and haemophilus polysaccharides, and tetanus and diphtheria toxoids using a 19-plexed assay.

    PubMed

    Whitelegg, Alison M E; Birtwistle, Jane; Richter, Alex; Campbell, John P; Turner, James E; Ahmed, Tarana M; Giles, Lynda J; Fellows, Mark; Plant, Tim; Ferraro, Alastair J; Cobbold, Mark; Drayson, Mark T; MacLennan, Calman A

    2012-03-30

    The measurement of antibody responses to vaccination is useful in the assessment of immune status in suspected immune deficiency. Previous reliance on enzyme-linked immunoabsorbent assays (ELISA) has been cumbersome, time-consuming and expensive. The availability of flow cytometry systems has led to the development of multiplexed assays enabling simultaneous measurement of antibodies to several antigens. We optimized a flow cytometric bead-based assay to measure IgG and IgM concentrations in serum to 19 antigens contained in groups of bacterial subunit vaccines: pneumococcal vaccines, meningococcal vaccines, Haemophilus influenzae b (Hib), and tetanus and diphtheria toxoid vaccines. 89-SF was employed as the standard serum. The assay was used to determine specific antibody levels in serum from 193 healthy adult donors. IgG and pneumococcal IgM antibody concentrations were measurable across 3 log10 ranges encompassing the threshold protective IgG antibody levels for each antigen. There was little interference between antibody measurements by the 19-plexed assay compared with monoplexed assays, and a lack of cross-reactive IgG antibody, but evidence for cross-reacting IgM antibody for 3/19 pneumococcal antigens. 90th centile values for 15/19 IgG concentrations and 12/12 IgM concentrations of the 193 adult sera were within these ranges and percentages of sera containing protective IgG antibody levels varied from 4% to 95% depending on antigen. This multiplexed assay can simultaneously measure antibody levels to 19 bacterial vaccine antigens. It is suitable for use in standard clinical practice to assess the in vivo immune response to test vaccinations and measure absolute antibody levels to these antigens.

  13. Diphtheria

    MedlinePlus

    ... breathing sound (stridor) Skin sores (usually seen in tropical areas) Sometimes there are no symptoms. Exams and ... tetanus-diphtheria ( Td ). (The shot also has vaccine medicine for an infection called tetanus .) If you have ...

  14. Licensure of a Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed and Inactivated Poliovirus Vaccine and Guidance for Use as a Booster Dose.

    PubMed

    Liang, Jennifer; Wallace, Greg; Mootrey, Gina

    2015-09-04

    On March 24, 2015, the Food and Drug Administration licensed an additional combined diphtheria and tetanus toxoids and acellular pertussis adsorbed (DTaP) and inactivated poliovirus (IPV) vaccine (DTaP-IPV) (Quadracel, Sanofi Pasteur Inc.). Quadracel is the second DTaP-IPV vaccine to be licensed for use among children aged 4 through 6 years in the United States (1). Quadracel is approved for administration as a fifth dose in the DTaP series and as a fourth or fifth dose in the IPV series in children aged 4 through 6 years who have received 4 doses of DTaP-IPV-Hib (Pentacel, Sanofi Pasteur) and/or DTaP (Daptacel, Sanofi Pasteur) vaccine (2,3). This report summarizes the indications for Quadracel vaccine and provides guidance from the Advisory Committee on Immunization Practices (ACIP) for its use.

  15. Immunogenicity and reactogenicity of diphtheria, tetanus and pertussis toxoids combined with inactivated polio vaccine, when administered concomitantly with or as a diluent for a Hib conjugate vaccine.

    PubMed

    Knutsson, N; Trollfors, B; Taranger, J; Bergfors, E; Sundh, V; Lagergård, T; Ostergaard, E; Cicirello, H; Käyhty, H

    2001-08-14

    In an open trial, 400 infants were randomized to vaccination with a combined diphtheria-tetanus-pertussis-inactivated polio vaccine (DTaP-IPV) either mixed with a Haemophilus influenzae type b (Hib) tetanus toxoid conjugate immediately before injection (DTaP-IPV/Hib (mix)) or given concurrently with the Hib conjugate at separate injection sites (DTaP-IPV+Hib (sep)). The pertussis component consisted of pertussis toxoid alone. The vaccines were given intramuscularly at 3, 5 and 12 months of age. No vaccine-related serious adverse events occurred. Local reactions were evaluated from diary cards completed by the parents. Infants who received DTaP-IPV/Hib (mix) experienced fewer local reactions. Sera were obtained 28-45 days after the second and third vaccinations. Total Hib capsular antibodies were similar in the two groups after the second injection but lower in the group receiving DTaP-IPV/Hib (mix) than in the group receiving DTaP-IPV+Hib (sep) after the third injection (geometric mean 6.1 vs 10.4 microg/ml). Mixing of the vaccines also led to somewhat lower diphtheria toxin antibodies (5.9 vs. 7.7 IU/ml after the third injection) while tetanus antibodies were higher (3.9 vs. 2.5 IU/ml after the third injection). Antibodies against pertussis toxin and the three polio virus types were similar in the two groups. The moderate impairment of the Hib antibody response caused by mixing of the Hib conjugate with aluminium adsorbed DTaP may be due to physicochemical interference but is probably of little clinical importance because of the ability of the Hib conjugates to induce an immunologic memory.

  16. Carrier priming with CRM 197 or diphtheria toxoid has a different impact on the immunogenicity of the respective glycoconjugates: biophysical and immunochemical interpretation.

    PubMed

    Pecetta, S; Lo Surdo, P; Tontini, M; Proietti, D; Zambonelli, C; Bottomley, M J; Biagini, M; Berti, F; Costantino, P; Romano, M R

    2015-01-03

    Glycoconjugate vaccines play an enormous role in preventing infectious diseases. The main carrier proteins used in commercial conjugate vaccines are the non-toxic mutant of diphtheria toxin (CRM197), diphtheria toxoid (DT) and tetanus toxoid (TT). Modern childhood routine vaccination schedules include the administration of several vaccines simultaneously or in close sequence, increasing the concern that the repeated exposure to conjugates based on these carrier proteins might interfere with the anti-polysaccharide response. Extending previous observations we show here that priming mice with CRM197 or DT does not suppress the response to the carbohydrate moiety of CRM197 meningococcal serogroup A (MenA) conjugates, while priming with DT can suppress the response to DT-MenA conjugates. To explain these findings we made use of biophysical and immunochemical techniques applied mainly to MenA conjugates. Differential scanning calorimetry and circular dichroism data revealed that the CRM197 structure was altered by the chemical conjugation, while DT and the formaldehyde-treated form of CRM197 were less impacted, depending on the degree of glycosylation. Investigating the binding and avidity properties of IgGs induced in mice by non-conjugated carriers, we found that CRM197 induced low levels of anti-carrier antibodies, with decreased avidity for its MenA conjugates and poor binding to DT and respective MenA conjugates. In contrast, DT induced high antibody titers able to bind with comparable avidity both the protein and its conjugates but showing very low avidity for CRM197 and related conjugates. The low intrinsic immunogenicity of CRM197 as compared to DT, the structural modifications induced by glycoconjugation and detoxification processes, resulting in conformational changes in CRM197 and DT epitopes with consequent alteration of the antibody recognition and avidity, might explain the different behavior of CRM197 and DT in a carrier priming context.

  17. Immunogenicity of MenACWY-CRM in Korean Military Recruits: Influence of Tetanus-Diphtheria Toxoid Vaccination on the Vaccine Response to MenACWY-CRM

    PubMed Central

    Kim, Han Wool; Park, In Ho; You, Sooseong; Yu, Hee Tae; Oh, In Soo; Sung, Pil Soo

    2016-01-01

    The quadrivalent meningococcal conjugate vaccine (MenACWY-CRM) has been introduced for military recruits in Korea since 2012. This study was performed to evaluate the immunogenicity of MenACWY-CRM in Korean military recruits. In addition, the influence of tetanus-diphtheria toxoids (Td) vaccination on the vaccine response to MenACWY-CRM was analyzed. A total of 75 military recruits were enrolled. Among them, 18 received a dose of MenACWY-CRM only (group 1), and 57 received Td three days before MenACWY-CRM immunization (group 2). The immunogenicity of MenACWY-CRM was compared between the two groups. The serum bactericidal activity with baby rabbit complement was measured before and three weeks after immunization against serogroups A, C, W-135, and Y. The geometric mean titers (GMTs) against four serogroups were significantly increased in both groups after immunization. Compared to group 2, group 1 exhibited significantly higher vaccine responses in several aspects: post-immune GMTs against serogroup A and C, seroresponse rates against serogroup A, and a fold increases of titers against serogroup A, C, and Y. MenACWY-CRM was immunogenic against all vaccine-serogroups in Korean military recruits. Vaccine response to MenACWY-CRM was influenced by Td administered three days earlier. PMID:27593883

  18. Modulation of benzo[a]pyrene induced neurotoxicity in female mice actively immunized with a B[a]P-diphtheria toxoid conjugate.

    PubMed

    Schellenberger, Mario T; Grova, Nathalie; Farinelle, Sophie; Willième, Stéphanie; Schroeder, Henri; Muller, Claude P

    2013-09-01

    Benzo[a]pyrene (B[a]P) is a small molecular weight carcinogen and the prototype of polycyclic aromatic hydrocarbons (PAHs). While these compounds are primarily known for their carcinogenicity, B[a]P and its metabolites are also neurotoxic for mammalian species. To develop a prophylactic immune strategy against detrimental effects of B[a]P, female Balb/c mice immunized with a B[a]P-diphtheria toxoid (B[a]P-DT) conjugate vaccine were sub-acutely exposed to 2mg/kg B[a]P and behavioral performances were monitored in tests related to learning and memory, anxiety and motor coordination. mRNA expression of the NMDA receptor (NR1, 2A and 2B subunits) involved in the above behavioral functions was measured in 5 brain regions. B[a]P induced NMDA1 expression in three (hippocampus, amygdala and cerebellum) of five brain regions investigated, and modulated NMDA2 in two of the five brain regions (frontal cortex and cerebellum). Each one of these B[a]P-effects was reversed in mice that were immunized against this PAH, with measurable consequences on behavior such as anxiety, short term learning and memory. Thus active immunization against B[a]P with a B[a]P-DT conjugate vaccine had a protective effect and attenuated the pharmacological and neurotoxic effects even of high concentrations of B[a]P.

  19. Immunogenicity of MenACWY-CRM in Korean Military Recruits: Influence of Tetanus-Diphtheria Toxoid Vaccination on the Vaccine Response to MenACWY-CRM.

    PubMed

    Kim, Han Wool; Park, In Ho; You, Sooseong; Yu, Hee Tae; Oh, In Soo; Sung, Pil Soo; Shin, Eui Cheol; Kim, Kyung Hyo

    2016-11-01

    The quadrivalent meningococcal conjugate vaccine (MenACWY-CRM) has been introduced for military recruits in Korea since 2012. This study was performed to evaluate the immunogenicity of MenACWY-CRM in Korean military recruits. In addition, the influence of tetanus-diphtheria toxoids (Td) vaccination on the vaccine response to MenACWY-CRM was analyzed. A total of 75 military recruits were enrolled. Among them, 18 received a dose of MenACWY-CRM only (group 1), and 57 received Td three days before MenACWY-CRM immunization (group 2). The immunogenicity of MenACWY-CRM was compared between the two groups. The serum bactericidal activity with baby rabbit complement was measured before and three weeks after immunization against serogroups A, C, W-135, and Y. The geometric mean titers (GMTs) against four serogroups were significantly increased in both groups after immunization. Compared to group 2, group 1 exhibited significantly higher vaccine responses in several aspects: post-immune GMTs against serogroup A and C, seroresponse rates against serogroup A, and a fold increases of titers against serogroup A, C, and Y. MenACWY-CRM was immunogenic against all vaccine-serogroups in Korean military recruits. Vaccine response to MenACWY-CRM was influenced by Td administered three days earlier.

  20. Modulation of benzo[a]pyrene induced neurotoxicity in female mice actively immunized with a B[a]P–diphtheria toxoid conjugate

    SciTech Connect

    Schellenberger, Mario T.; Grova, Nathalie; Farinelle, Sophie; Willième, Stéphanie; Muller, Claude P.

    2013-09-01

    Benzo[a]pyrene (B[a]P) is a small molecular weight carcinogen and the prototype of polycyclic aromatic hydrocarbons (PAHs). While these compounds are primarily known for their carcinogenicity, B[a]P and its metabolites are also neurotoxic for mammalian species. To develop a prophylactic immune strategy against detrimental effects of B[a]P, female Balb/c mice immunized with a B[a]P–diphtheria toxoid (B[a]P–DT) conjugate vaccine were sub-acutely exposed to 2 mg/kg B[a]P and behavioral performances were monitored in tests related to learning and memory, anxiety and motor coordination. mRNA expression of the NMDA receptor (NR1, 2A and 2B subunits) involved in the above behavioral functions was measured in 5 brain regions. B[a]P induced NMDA1 expression in three (hippocampus, amygdala and cerebellum) of five brain regions investigated, and modulated NMDA2 in two of the five brain regions (frontal cortex and cerebellum). Each one of these B[a]P-effects was reversed in mice that were immunized against this PAH, with measurable consequences on behavior such as anxiety, short term learning and memory. Thus active immunization against B[a]P with a B[a]P–DT conjugate vaccine had a protective effect and attenuated the pharmacological and neurotoxic effects even of high concentrations of B[a]P. - Highlights: • B[a]P-antibodies attenuated B[a]P induced NMDA expression in several brain regions. • B[a]P had measurable consequences on anxiety, short term learning and memory. • B[a]P immunization attenuated the pharmacological and neurotoxic effects of B[a]P. • Vaccination may also provide some protection against chemical carcinogenesis.

  1. Modulation of Benzo[a]pyrene induced immunotoxicity in mice actively immunized with a B[a]P-diphtheria toxoid conjugate

    SciTech Connect

    Schellenberger, Mario T.; Grova, Nathalie; Willieme, Stephanie; Farinelle, Sophie; Prodhomme, Emmanuel J.F.

    2009-10-01

    Benzo[a]pyrene (B[a]P) is a small molecular weight carcinogen and the prototype of polycyclic aromatic hydrocarbons (PAHs). While these compounds are primarily known for their carcinogenicity, B[a]P and its metabolites are also toxic for mammalian immune cells. To develop a prophylactic immune strategy against detrimental effects of B[a]P, we have immunized mice with a B[a]P-diphtheria toxoid conjugate vaccine. We showed that high levels of antibodies against B[a]P and its metabolites modulate the redistribution of these PAHs in the blood. After immunization, increased levels of B[a]P and its metabolites were recovered in the blood. B[a]P significantly suppressed the proliferative response of both T and B cells after a sub-acute administration, an effect that was completely reversed by vaccination. In immunized mice also the immunotoxic effect of B[a]P on IFN-{gamma}, IL-12, TNF-{alpha} production and the reduced B cell activation was restored. Finally, our results showed that specific antibodies inhibited the induction of Cyp1a1 by B[a]P in lymphocytes and Cyp1b1 in the liver, enzymes that are known to convert the procarcinogen B[a]P to the ultimate DNA-adduct forming metabolite, a major risk factor of chemical carcinogenesis. Thus, we demonstrate that vaccination with a B[a]P conjugate vaccine based on a carrier protein used in licensed human vaccines reduces immunotoxicity and possibly other detrimental effects associated with B[a]P.

  2. Evaluation of the Immunogenicity of Diphtheria Toxoid Conjugated to Salmonella Typhimurium-Derived OPS in a Mouse Model: A Potential Vaccine Candidate Against Salmonellosis

    PubMed Central

    Amini, Vahid; Kazemian, Hossein; Yamchi, Jalil Kardan; Feyisa, Seifu Gizaw; Aslani, Saeed; Shavalipour, Aref; Houri, Hamidreza; Hoorijani, Mohammadneshvan; Halaji, Mehrdad; Heidari, Hamid

    2016-01-01

    Background Salmonella enterica serovar Typhimurium (S. Typhimurium) causes gastroenteritis in humans and paratyphoid disease in some animals. Given the emergence of antibiotic resistance, vaccines are more effective than chemotherapy in disease control. Objectives The aim of this experimental study was to evaluate the immunogenicity of diphtheria toxoid (DT) conjugated with S. Typhimurium -derived OPS (O side chain isolation) in mice to determine its potential as a vaccine candidate against salmonellosis. Materials and Methods Lipopolysaccharide (LPS) was extracted from the bacterial strain. After isolation of the O side chain of LPS, detoxification, and conjugation of the detoxified OPS samples with DT, pyrogenicity, toxicity, and sterility tests were performed. To vaccination, four groups of female Balb/c mice were used in an immunization test. Antibody responses were measured by the ELISA method. Challenging processes were performed to analyze the efficacy of the OPS-DT compound. Results Two weeks after the first vaccination dose, there was no significant difference in the antibody titers of the OPS and OPS-DT groups. However, after the second and third doses, the antibody titers of the OPS-DT group increased significantly compared with those of the control groups (P < 0.001). The induction of anti-OPS antibodies was as follows: OPS-DT>OPS. The most anti-OPS IgG antibody was IgG1. Challenging procedure showed successful protective characteristics in clinical examinations. Conclusions The results indicated that DT increased anti-OPS antibodies against the OPS-DT compound. The antibody response to OPS-DT was greater than that to OPS alone. We conclude that OPS-DT is an appropriate and acceptable vaccine candidate against salmonellosis. PMID:27660722

  3. Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines - extended study: correlation of serology with in vivo toxin neutralisation.

    PubMed

    Sesardic, D; Winsnes, R; Rigsby, P; Behr-Gross, M-E

    2004-01-01

    Phase I of BSP034 collaborative study was extended in two laboratories to include correlation of serology with in vivo toxin neutralisation test (TNT) using 2 separate sets of 20 serum pools, produced in-house. The study investigated the extent to which the in vitro methods for diphtheria antibodies, Vero cell assay and diphtheria enzyme-linked immunosorbent assay for diphtheria antitoxin (D-ELISA), can detect neutralising antibodies by comparison with TNT in guinea pigs. The study was also performed to compare the antibody neutralising potency obtained in relation to guinea pig (GP) or equine (DI) antitoxin standard. In addition, the study provided an opportunity to compare ELISA for tetanus antitoxin (T-ELISA) and TNT assay for detection of anti-tetanus antibodies, from the same set of serum pools. The data obtained show that antitoxin potency obtained by Vero cell assay, D-ELISA and T-ELISA using the same GP standard, highly correlated with neutralising potency as determined in respective TNT assays. Vero cell assay with DI provided estimates that also correlated with neutralising potency, but were of significantly lower titre. Since reference to DI standard is widely used in serodiagnosis, as well as in clinical studies where diphtheria antitoxin titres obtained in the Vero cell method are taken as surrogate markers for vaccine efficacy, it should be investigated if a similar difference is also observed for human serology.

  4. Carrier priming effect of CRM197 is related to an enhanced B and T cell activation in meningococcal serogroup A conjugate vaccination. Immunological comparison between CRM197 and diphtheria toxoid.

    PubMed

    Pecetta, S; Tontini, M; Faenzi, E; Cioncada, R; Proietti, D; Seubert, A; Nuti, S; Berti, F; Romano, M R

    2016-04-29

    Glycoconjugate vaccines are composed of capsular polysaccharides (CPSs) of a pathogenic bacteria covalently linked to carrier proteins. Pre-exposure to the carrier is known to influence the efficacy of the glycoconjugate, by inducing enhanced or suppressed anti-CPS response. Following our previous work on the immunogenicity of diphtheria toxin mutant CRM197 and formaldehyde-treated diphtheria toxoid (DT) as carriers for meningococcal A (MenA) conjugates in mouse model, we further investigated the role of the carrier on the immunological response to glycoconjugate vaccines. We previously showed that high dosage DT priming could result in carrier-induced epitopic suppression (CIES), an event that did not occur for CRM197 priming, and we observed that anti-DT IgGs could cross-react with DT based conjugates in vitro. Here, we confirmed the cross-reactivity of anti-carrier IgGs with DT conjugates in vivo. Furthermore, we analyzed the splenocytes of animals primed with the carrier and subsequently immunized with the MenA conjugate. Pre-exposure to the carrier protein, both CRM197 and DT, resulted in increased carrier-specific plasma and memory B cell response. However, only for CRM197 priming an enhanced carbohydrate-specific plasma cell response was observed. Analysis of circulating IgGs confirmed these observations. Memory to the CPS resulted to be non-influenced by carrier priming. Analysis of T helper response showed an enhancement effect for CRM197 priming, while DT priming resulted in constrained T cell activation. Stimulation with CRM197, which does not require formaldehyde detoxification, of splenocytes from animal immunized with DT suggested that the formaldehyde treatment used to produce DT might be the cause of limited presentation of the antigen to the T cells. We concluded that the dominant carrier-specific B cell response in case of limited T cell recruitment might explain the previously observed CIES phenomenon in case of DT priming.

  5. Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin

    PubMed Central

    Avila-Alonso, Ana; González-Rivera, Milagros; Tamayo, Eduardo; Eiros, Jose María; Almansa, Raquel

    2016-01-01

    Diphtheria antitoxin for therapeutic use is in limited supply. A potential source might be affinity-purified antibodies originally derived from plasma of adults who received a booster dose of a vaccine containing diphtheria toxoid. These antibodies might be useful for treating even severe cases of diphtheria. PMID:27314309

  6. Safety and immunogenicity of a single intramuscular dose of a tetanus-diphtheria toxoid (Td) vaccine (BR-TD-1001) in healthy Korean adult subjects.

    PubMed

    Hong, Taegon; Chung, Yong-Ju; Kim, Tae-Yeon; Kim, Ik-Hwan; Choe, Yong-Kyung; Lee, Jongtae; Jeon, Sangil; Han, Seunghoon; Yim, Dong-Seok

    2015-01-01

    BR-TD-1001 was developed as a booster for the immunity maintenance of diphtheria and tetanus. The aim of this study was to evaluate the safety and immunogenicity of BR-TD-1001 (test vaccine) in comparison with placebo and an active comparator in healthy Korean adults. A randomized, double-blind, placebo-controlled, active comparator, phase I clinical trial was conducted. Fifty subjects were randomly assigned to one of 3 treatment groups in a ratio of 2:2:1, and were administered a single intramuscular dose of test vaccine, active comparator, or placebo, respectively. All subjects were monitored for 4 weeks after injection. The antibody titers of the patients 2 and 4 weeks after vaccination were compared with the baseline. The frequencies of all adverse events including adverse drug reactions in the test group were not statistically different from those of the other treatment groups (P = 0.4974, 0.3061). No serious adverse event occurred, and no subject was withdrawn from the study for safety. The seroprotection rates against both tetanus and diphtheria at 4 weeks after vaccination were over 0.95. For anti-tetanus antibody, the geometric mean titer in the test group was significantly higher than those of the other groups (P = 0.0364, 0.0033). The geometric mean titer of anti-diphtheria antibody in the test group was significantly higher than the value of the placebo (P = 0.0347) while it was not for the value of the active comparator (P = 0.8484). In conclusion, BR-TD-1001 was safe, well-tolerated, and showed sufficient immunogenicity as a booster for diphtheria and tetanus.

  7. Suppression and modulation of cellular and humoral immune responses to Haemophilus influenzae type B (Hib) conjugate vaccine in hib-diphtheria-tetanus toxoids-acellular pertussis combination vaccines: a study in a rat model.

    PubMed

    Mawas, Fatme; Newman, Gareth; Burns, Samantha; Corbel, Michael J

    2005-01-01

    We assessed a rat model to evaluate the immunogenicity of Haemophilus influenzae type b (Hib) conjugate vaccines and the effect on Hib immunogenicity of combining 2 Hib vaccines (Hib-tetanus toxoid [TT]-A and Hib-TT-B) with diphtheria-TT-acellular pertussis (DTaP)(3) or DTaP(5)/inactivated poliovirus (IPV) vaccines. Rats were immunized subcutaneously with Hib alone or with Hib and DTaP-based vaccines; anti-Hib capsular polysaccharide IgG, poly-ribosyl-ribitol-phosphate (PRP), IgG subclass, and cellular immune responses were evaluated. Results showed a significant reduction in the antibody response to PRP when Hib-TT-A was administered in combination with DTaP(3) and showed changes in the anti-PRP IgG subclass distribution between the separate and combination groups. However, combining Hib-TT-B with DTaP(5)/IPV did not reduce the anti-PRP antibody response. These results suggest that the model can predict the effect of combined administration of Hib and DTaP vaccines on Hib immunogenicity and would be suitable for preclinical studies of mechanisms of interference in Hib/DTaP vaccines.

  8. Comparison of the Safety and Immunogenicity of a Novel Quadrivalent Meningococcal ACWY-Tetanus Toxoid Conjugate Vaccine and a Marketed Quadrivalent Meningococcal ACWY-Diphtheria Toxoid Conjugate Vaccine in Healthy Individuals 10–25 Years of Age

    PubMed Central

    Halperin, Scott A.; Baine, Yaela; Domachowske, Joseph B.; Aggarwal, Naresh; Simon, Michael; Langley, Joanne M.; McNeil, Shelly A.; Friedland, Leonard R.; Bianco, Veronique; Baccarini, Carmen I.; Miller, Jacqueline M.

    2014-01-01

    Background Universal immunization of adolescents against meningococcal disease with a quadrivalent meningococcal ACWY (MenACWY) conjugate vaccine is recommended in a number of countries. Methods In a randomized, controlled, observer-blinded, multicenter trial, 1016 participants, 10–25 years of age, were randomly allocated 1:1:1 to receive a single dose of 1 of 2 lots of an investigational tetanus toxoid‐conjugated MenACWY vaccine (MenACWY‐TT) or a marketed diphtheria toxoid‐conjugated MenACWY vaccine (MenACWY‐DT). The primary outcome was the noninferiority of the vaccine response after MenACWY‐TT (lot A) compared with MenACWY‐DT for all 4 serogroups. Vaccine response was defined as a postvaccination human serum bactericidal antibody (hSBA) titer against each of the serogroups of at least 1:8 in persons initially seronegative (<1:4) or as a 4‐fold increase in titer pre‐ to postvaccination in persons initially seropositive (≥1:4). Adverse events (AEs) after immunization were measured 4 and 31 days postvaccination. Results The mean age of participants was 16.3 years; 977 (96.6%) completed the study. The noninferiority of MenACWY‐TT (lot A) to the control vaccine in terms of the percentage of participants with hSBA vaccine response was demonstrated for each serogroup. Vaccine response rates ranged from 51.0% to 82.5% for the 4 serogroups after MenACWY‐TT (both lots) compared with 39.0%–76.3% for the 4 serogroups after MenACWY‐DT. Pain was the most common injection‐site reaction reported by 50.8%–55.4% across the 3 groups. Fatigue and headache were the most common systemic solicited AEs, reported by 27.3%–29.2% and 25.5%–26.4%, respectively. Conclusions Tetanus toxoid‐conjugated MenACWY vaccine was well tolerated and elicited an immune response that was noninferior to that of a marketed MenACWY‐DT (www.clinicaltrials.gov NCT01165242). PMID:24567843

  9. Immunogenicity and safety of four different doses of Haemophilus influenzae type b-tetanus toxoid conjugated vaccine, combined with diphtheria-tetanus-pertussis vaccine (DTP-Hib), in Indonesian infants.

    PubMed

    Punjabi, Narain H; Richie, Emily L; Simanjuntak, Cyrus H; Harjanto, Sri Juliani; Wangsasaputra, Ferry; Arjoso, Sumarjati; Rofiq, Ainur; Prijanto, Mulyati; Julitasari; Yela, Ursula; Herzog, Christian; Cryz, Stanley J

    2006-03-10

    Widespread use of Haemophilus influenzae type b (Hib) conjugated vaccine in industrialized countries has resulted in a dramatic decline in the incidence of invasive Hib diseases, but the vaccine's cost has prevented its inclusion in basic immunization programs in developing countries. To overcome this problem, combination with diphtheria-tetanus-pertussis (DTP) vaccine or reduction in the dose of Hib vaccine has been proposed. To evaluate the immunogenicity and adverse reactions from lower doses of Hib-polyribosylphosphate (PRP) conjugated with tetanus toxoid (PRP-T), a double-blind study was conducted in Jakarta, Indonesia, and its suburbs. A total of 1048 infants 6 weeks to 6 months of age received three doses of DTP vaccine combined with the usual 10 microg dose or with a reduced dose of 5, 2.5 or 1.25 microg of PRP-T at two-monthly intervals. Antibodies were measured prior to the first dose and 4-6 weeks following the third dose. Adverse reactions were similar among all four groups. The only significant difference was a higher rate of irritability (p<0.02) and of temperature elevation >38 degrees C (p<0.009) after doses 1 and 2 in the lowest dose group (1.25 microg PRP-T) compared to the other groups. All participants tested had a 4-fold increase in antibodies against all DTP antigens. In addition, after a fourth booster dose of Hib, 99.6% of infants produced >or=0.15 microg/ml of antibody to Hib-PRP, and 96.4% showed levels >or=1.0 microg/ml after primary immunization, level that correlate with short- and long-term immunity, respectively. Antibody titers to the PRP antigen showed no significant differences among dosage groups with the exception of the 5.0 microg group, which had a significantly higher GMC than the 1.25 microg group (p<0.012). This study demonstrates that primary vaccination with half, one-fourth, or one-eighth of the usual dose of PRP-T, combined with DTP vaccine, produces protective immune responses, and has side effects that are comparable

  10. [Study of diphtheria anatoxins in immunochemical and tissue culture assays].

    PubMed

    Gal'vidis, I A; Sviridov, V V

    2007-01-01

    Equine diphtheria antitoxins from different manufacturers were studied. Their immunochemical interaction with diphtheria toxin, toxoid, and antigens of Corynebacterium diphtheriae in ELISA and immunoblotting assays as well as biological activity in CHO cell assay were compared. The discovered differences between antitoxin samples with stated equal activity in IU/ml point to heterogeneity of antigen composition in preparations used for immunization. Mentioned methods allow to standardize antitoxins basing on their biological activity and immunochemical characteristics.

  11. Diphtheria immunity status in Egypt.

    PubMed

    Redwan, El-Rashdy M; El-Awady, Mostafa K

    2004-01-01

    The aim of this study was to determine immune status to corynebacterium diphtheria by screening for protective antibodies in a sample of Egyptian population. The study population consisted of 709 healthy subjects aged from 2 months to 105 years, inhabitants of 6 regions of Egypt. The study utilized Enzyme linked immunosorbent assay (ELISA) to measure serum levels IgG antibodies reactive with diphtheria toxoid. Levels of diphtheria toxoid antibody > or = 0.1 IU/ ml were defined as immune/protected, 23.9 % of the population were found to be susceptible to diphtheria (IgG level < 0.01 IU/ml), 43% had basic protection (0.01-0.09 IU/ml), and 33.1% were fully protected (0.1 IU/ml). The results revealed that serum levels of antitoxin antibodies decreased in old ages (< 60 y) with the females being more susceptible then males. These results recommend a booster immunization for the susceptible age groups.

  12. Randomized, controlled, multicenter study of the immunogenicity and safety of a fully liquid combination diphtheria-tetanus toxoid-five-component acellular pertussis (DTaP5), inactivated poliovirus (IPV), and haemophilus influenzae type b (Hib) vaccine compared with a DTaP3-IPV/Hib vaccine administered at 3, 5, and 12 months of age.

    PubMed

    Vesikari, Timo; Silfverdal, Sven Arne; Boisnard, Florence; Thomas, Stéphane; Mwawasi, Grace; Reynolds, Donna

    2013-10-01

    This study compared the levels of immunogenicity and safety of diphtheria-tetanus toxoid-five-component acellular pertussis (DTaP(5)), inactivated poliovirus (IPV), and Haemophilus influenzae type b (Hib) (DTaP(5)-IPV-Hib) and DTaP(3)-IPV/Hib vaccines for study participants 3, 5, and 12 months of age. Post-dose 3 noninferiority criteria comparing DTaP(5)-IPV-Hib to DTaP(3)-IPV/Hib using rates of seroprotection were demonstrated against diphtheria, tetanus, and polio types 1 to 3, but not for polyribosylribitol phosphate (PRP). While PRP did not meet noninferiority criteria, the seroprotection rate and geometric mean concentration (GMC) were high, indicating a clinically robust immune response. GMCs or titers for other antigens (including pertussis) and the safety profiles were generally similar between groups. Fully liquid DTaP(5)-IPV-Hib can be administered using the 3-, 5-, and 12-month vaccination schedule. (This study has been registered at ClinicalTrials.gov under registration no. NCT00287092.).

  13. Purification of Clostridium toxoids.

    PubMed

    Buchowicz, I; Hay, M; Schiller, B; Korbecki, M; Sochańska, R

    1977-01-01

    A two-step fractionation procedure was applied for purification and concentration of the individual Clostridium toxoids. The toxoids were precipitated with hydrochloric acid in the presence of sodium sextametaphosphate, then antigenic fractions were separated from inactive contaminants by Sephadex G-75 filtration. Specific activity of the preparations thus obtained, as determined by Mancini radial immunodiffusion, was 150--565 binding units per mg of protein nitrogen for Clostridium perfringens toxoid, 204--352 binding units for Clostridium oedematiens toxoid and 26.6 -- 51.2 binding units for Clostridium septicum toxoid.

  14. Diphtheria Photos

    MedlinePlus

    ... Whooping cough Influenza (flu) Rabies Yellow fever Diphtheria Photos Photographs accompanied by text that reads "Courtesy of . . . " ... source (not IAC) for permission to reprint copyrighted photos. Warning : Some of these photos are quite graphic. ...

  15. Diphtheria outbreak in Thailand, 2012; seroprevalence of diphtheria antibodies among Thai adults and its implications for immunization programs.

    PubMed

    Wanlapakorn, Nasamon; Yoocharoen, Pornsak; Tharmaphornpilas, Piyanit; Theamboonlers, Apiradee; Poovorawan, Yong

    2014-09-01

    An age distribution shift in diphtheria cases during a 2012 outbreak in northeastern of Thailand suggests adults are increasingly at risk for infection in Thailand. Data regarding immunity against diphtheria among the adult Thai population is limited. We review a 2012 diphtheria outbreak in Thailand and conducted a nationwide seroepidemiological survey to determine the prevalence of diphtheria antibodies among Thai adults in order to inform immunization programs. A total of 41 confirmed cases, 6 probable cases and 101 carriers of diphtheria were reported from northeastern and upper southern Thailand. The diphtheria outbreak in northeastern Thailand occurred among adults aged > or =15 years; sporadic cases occurred among children from upper southern Thailand. We conducted a seroepidemiological survey of 890 Thai adults from 4 age groups (20-29, 30-39, 40-49 and 50-59 years) in 7 different geographical areas of Thailand (Chiang Mai, Ratchaburi, Chon Buri, Nakhon Si Thammarat, Phitsanulok, Khon Kaen and Songkhla). Diptheria toxin antibody levels were measured with a commercially available ELISA test. The seroprotection rate ranged from 83% to 99%, with the highest in eastern Thailand (Chon Buri, 99%) and the lowest in northern Thailand (Chiang Mai, 83%). Diphtheria antibodies declined with increasing age. We recommend one doseof diphtheria-tetanus toxoid (dT) vaccine once after 20 years of age in order to boost the antibody and revaccinations every 10 years to prevent future outbreaks.

  16. Identification of a human monoclonal antibody to replace equine diphtheria antitoxin for treatment of diphtheria intoxication.

    PubMed

    Sevigny, Leila M; Booth, Brian J; Rowley, Kirk J; Leav, Brett A; Cheslock, Peter S; Garrity, Kerry A; Sloan, Susan E; Thomas, William; Babcock, Gregory J; Wang, Yang

    2013-11-01

    Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 μg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.

  17. Comparison of four serological methods for the detection of diphtheria anti-toxin antibody.

    PubMed

    Walory, J; Grzesiowski, P; Hryniewicz, W

    2000-11-01

    The aim of this study was to compare four serological methods for the detection of Corynebacterium diphtheriae IgG anti-toxin antibodies (IgG-DTAb) in human serum. One hundred serum samples were evaluated for C. diphtheriae IgG-DTAb by four different methods: passive haemagglutination (PHA), latex agglutination test (LA), toxoid enzyme-linked immunosorbent assay (Toxoid-ELISA), and toxin-binding inhibition enzyme-linked immunosorbent assay (ToBI-ELISA). As the external standardisation the neutralisation test for C. diphtheriae toxin in Vero cells (TN Vero) was used. For internal standardisation of IgG-DTAb titres, the WHO standard serum of human diphtheria antitoxin was used. The study revealed a poor correlation between the reference test and the PHA (r=0.34 Pearson's correlation coefficient), an acceptable correlation for the LA (r=0.74), a good correlation for the Toxoid-ELISA (r=0.81) and a very good correlation for ToBI-ELISA (r=0.93). The sensitivity measurements of PHA, LA, Toxoid-ELISA and ToBI-ELISA tests, were 14, 100, 94, 96% respectively and the corresponding specificity characteristics were 86, 76, 94, 90 respectively. Of the four evaluated methods, the ToBI-ELISA could be recommended for scientific and precise laboratory assays of diphtheria antibody levels in humans. For screening purposes the Toxoid-ELISA could be used, but the accuracy of antibody titres below 0.1 IU/ml, considered as the limits of protection, is questionable. Both tests offer very useful alternatives to the in vitro diphtheria toxin neutralisation test in Vero cells. Because of their unsatisfactory correlation and sensitivity as compared to the reference method, PHA and LA should be avoided and replaced by one of the two enzyme immunoassays.

  18. Diphtheria Disease Villain

    MedlinePlus

    ... disease villain from BAM! Body and Mind . Case file: tissue trolls Real name: diphtheria Known aliases: Corynebacterium ... Action Coalition (IAC) Diphtheria and the Alaskan Iditarod File Formats Help: How do I view different file ...

  19. [The return of diphtheria in Europe. Is the French population protected?].

    PubMed

    Rey, M; Vincent-Ballereau, F; Patey, O

    1997-01-01

    Following the generalized vaccination of children, the European countries achieved the elimination of diphtheria. However the huge epidemic which rages since 1990 in the New Independent States of ex-URSS, culminating in 1994-1995 (with respectively 47,802 and 50,412 notified cases), has showed that diphtheria could be still threatening. A serosurvey was carried out in France on 1,025 adult patients attending the emergency wards of three hospitals, located in different parts of this country. This study showed the insufficient immunity of adults, for lack of a routine programme for revaccination: only half of them have antibodies assuring their protection. The antitoxic immunity decreases according to age. This decline is more marked in women than in men, most of them being reimmunized during the military service. These data confirm the alarming results of similar serosurveys performed in others industrialized countries. The present risk of importing diphtheria in these countries requires a strengthening of clinical and bacteriological surveillance of all cases of infections attributable to C. diphtheriae, and could justify a revaccination programme for adult population, including a regular booster, at 10 year-interval, of a reduced dose of diphtheria toxoid (d), and/or using the combined toxoid Td in the wounded, instead of tetanus toxoid alone.

  20. Pangenomic study of Corynebacterium diphtheriae that provides insights into the genomic diversity of pathogenic isolates from cases of classical diphtheria, endocarditis, and pneumonia.

    PubMed

    Trost, Eva; Blom, Jochen; Soares, Siomar de Castro; Huang, I-Hsiu; Al-Dilaimi, Arwa; Schröder, Jasmin; Jaenicke, Sebastian; Dorella, Fernanda A; Rocha, Flavia S; Miyoshi, Anderson; Azevedo, Vasco; Schneider, Maria P; Silva, Artur; Camello, Thereza C; Sabbadini, Priscila S; Santos, Cíntia S; Santos, Louisy S; Hirata, Raphael; Mattos-Guaraldi, Ana L; Efstratiou, Androulla; Schmitt, Michael P; Ton-That, Hung; Tauch, Andreas

    2012-06-01

    Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ω(tox)(+) phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox(+) corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.

  1. 9 CFR 113.114 - Tetanus Toxoid.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Tetanus Toxoid. 113.114 Section 113... Bacterial Products § 113.114 Tetanus Toxoid. Tetanus Toxoid shall be produced from a culture of Clostridium... purified and concentrated. Each serial of biological product containing tetanus toxoid fraction shall...

  2. 9 CFR 113.114 - Tetanus Toxoid.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Tetanus Toxoid. 113.114 Section 113... Bacterial Products § 113.114 Tetanus Toxoid. Tetanus Toxoid shall be produced from a culture of Clostridium... purified and concentrated. Each serial of biological product containing tetanus toxoid fraction shall...

  3. Diphtheria epidemic in the Republic of Georgia, 1993-1997.

    PubMed

    Khetsuriani, N; Imnadze, P; Dekanosidze, N

    2000-02-01

    Epidemic diphtheria reemerged in the republic of Georgia in 1993. From 1993 to 1997, 1405 cases were reported (28 in 1993, 312 in 1994, 429 in 1995, 348 in 1996, and 288 in 1997), with a cumulative incidence of 25.8/100,000 and a case fatality ratio of 9.5%. During 1993-1997, 53% of the diphtheria cases occurred among persons >/=15 years of age. Unvaccinated patients were more likely to have toxic forms (relative risk=2.24; 95% confidence interval=1.69-2.96) or to die of diphtheria (relative risk=2.24; 95% confidence interval=1. 36-3.68) than those who had received at least one dose of diphtheria toxoid. Improvement in routine childhood vaccination coverage and implementation of mass adult vaccination campaigns have been critical to bringing the epidemic under control. By mid-1998, the overall diphtheria situation in Georgia appeared to have been controlled. Only 53 cases were reported from January to June 1998, representing a 64% decrease from the 148 cases during the corresponding period in 1997.

  4. Diphtheria (For Parents)

    MedlinePlus

    ... pregnancy Although most children tolerate it well, the vaccine sometimes causes mild side effects such as redness or tenderness at the injection ... throat cultures, and booster doses of the diphtheria vaccine. They will also receive ... have taken effect, someone with diphtheria will need bed rest for ...

  5. Evolution, epidemiology and diversity of Corynebacterium diphtheriae: New perspectives on an old foe.

    PubMed

    Sangal, Vartul; Hoskisson, Paul A

    2016-09-01

    Diphtheria is a debilitating disease caused by toxigenic Corynebacterium diphtheriae strains and has been effectively controlled by the toxoid vaccine, yet several recent outbreaks have been reported across the globe. Moreover, non-toxigenic C. diphtheriae strains are emerging as a major global health concern by causing severe pharyngitis and tonsillitis, endocarditis, septic arthritis and osteomyelitis. Molecular epidemiological investigations suggest the existence of outbreak-associated clones with multiple genotypes circulating around the world. Evolution and pathogenesis appears to be driven by recombination as major virulence factors, including the tox gene and pilus gene clusters, are found within genomic islands that appear to be mobile between strains. The number of pilus gene clusters and variation introduced by gain or loss of gene function correlate with the variable adhesive and invasive properties of C. diphtheriae strains. Genomic variation does not support the separation of C. diphtheriae strains into biovars which correlates well with findings of studies based on multilocus sequence typing. Genomic analyses of a relatively small number of strains also revealed a recombination driven diversification of strains within a sequence type and indicate a wider diversity among C. diphtheriae strains than previously appreciated. This suggests that there is a need for increased effort from the scientific community to study C. diphtheriae to help understand the genomic diversity and pathogenicity within the population of this important human pathogen.

  6. Absence of protective immunity against diphtheria in a large proportion of young adults.

    PubMed

    Rappuoli, R; Podda, A; Giovannoni, F; Nencioni, L; Peragallo, M; Francolini, P

    1993-01-01

    The schedule of vaccination recommended worldwide for diphtheria, tetanus and other diseases, provides good immunity during childhood. However, little attention has been paid to effective immunity in adults. We have collected sera from 334 Italian Army recruits and tested them for the presence of protective immunity against diphtheria and tetanus. In vivo neutralization assays were performed on rabbits and mice and values below 1/100 IU ml-1 were considered negative. Of the recruits, 22.9% were negative for diphtheria, while only 5.3% had no protective immunity against tetanus. This finding shows that a large proportion of Italian young adults are susceptible to diphtheria, and this could be dangerous if they travel to sites where this disease is still endemic, or if they come into contact with people coming from such areas. A booster vaccination of young adults against diphtheria should become common practice to avoid this risk. To reduce the side effects which are often associated with diphtheria vaccination in adults, we have developed a vaccine which contains a highly purified, non-toxic mutant of diphtheria toxin. This vaccine is combined with tetanus toxoid and can be routinely used as a booster in adults.

  7. Quadracel: Vaccination Against Diphtheria, Tetanus, Pertussis, and Poliomyelitis in Children

    PubMed Central

    Mosley, Juan F.; Smith, Lillian L.; Parke, Crystal K.; Brown, Jamal A.; LaFrance, Justin M.; Clark, Patricia K.

    2016-01-01

    Introduction: Vaccinations in school-aged children are required by state and local law to maintain high vaccination coverage rates, as well as low rates of vaccine-preventable diseases. Diphtheria, tetanus, and pertussis are childhood diseases that can be life threatening; poliomyelitis, another childhood disease, can be disabling. In turn, vaccinations were developed to provide protection against these diseases. Today, several vaccinations are recommended for children, including but not limited to diphtheria, tetanus, and pertussis (DTaP) and poliomyelitis (IPV). DTaP requires five doses, and IPV requires four. Quadracel (diphtheria and tetanus toxoids and acellular pertussis adsorbed and inactivated poliovirus vaccine, Sanofi Pasteur Inc.) is a new vaccination developed to condense the last dose of both DTaP and IPV so they do not have to be given separately, thus reducing the total number of vaccinations required. Discussion: The Quadracel vaccine is an option for use in children who are completing the DTaP and IPV series. In a randomized, controlled, phase 3, pivotal trial, Quadracel proved to be as efficacious and safe as Daptacel (diphtheria and tetanus toxoids and acellular pertussis vaccine adsorbed, Sanofi Pasteur Inc.) and IPOL (poliovirus vaccine inactivated, Sanofi Pasteur Inc.), given separately, to children between the ages of 4 and 6 years. Conclusion: Quadracel should be recommended to parents who have children between the ages of 4 and 6 years who meet the necessary administration criteria and need to finalize their DTaP and IPV series. Quadracel’s administration in the vaccination series replaces one additional injection, which may benefit children who are afraid of receiving shots and parents who need to schedule one less doctor’s appointment. PMID:27069343

  8. [Development studies of lyophilized standard diphtheria-tetanus-pertussis vaccines].

    PubMed

    Ozcengiz, Erkan; Unver, Derya; Cayan, H Hüseyin; Atakan Ablay, Pinar; Kanik, Esin

    2007-04-01

    In this study, diphtheria, tetanus and pertussis vaccine components were prepared as the formulations of diphtheria-tetanus-pertussis (DTP), diphtheria-tetanus (DT) for children, diphtheria-tetanus (Td) for adults, and tetanus toxoid (TT), respectively. Alhydrogel-adsorbed vaccines prepared to contain the stabilizing substances were lyophilized and the immunogenicity tests were carried out both in vivo and in vitro. The potencies of the tetanus component of the vaccines were obtained by the lethal challenge test in mice. The values were found as 144.86 IU/ml for lyophilized adsorbed (LA)-DTP, 116.5 IU/ml for LA-DT, 98.25 IU/ml for LA-Td and 96.2 IU/ml for LA-TT. Anti-tetanus IgG and anti-diphteria IgG levels determined by ELISA method were found high in the sera taken from the mice immunized with the above-mentioned vaccines. Anti-B.pertussis fimbria IgG antibody levels were also high by both ELISA and microagglutination tests. The test preparations were then compared to adsorbed liquid vaccines and it was shown that the components were quite stable in the lyophilized formulations. It was concluded that the formulations prepared in this study can be used as standard vaccines after being calibrated against World Health Organization standards.

  9. Diphtheria (For Parents)

    MedlinePlus

    ... depends almost completely on giving the diphtheria/tetanus/pertussis vaccine to children (DTaP) and non-immunized adolescents ... For Kids For Parents MORE ON THIS TOPIC Whooping Cough (Pertussis) Strep Test: Rapid Strep Test: Throat Culture ...

  10. Recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant enhances induction of diphtheria and tetanus antitoxin antibodies in mice by intranasal administration with diphtheria-pertussis-tetanus (DPT) combination vaccine.

    PubMed

    Isaka, Masanori; Komiya, Takako; Takahashi, Motohide; Yasuda, Yoko; Taniguchi, Tooru; Zhao, Yanqiu; Matano, Keiko; Matsui, Hideyuki; Maeyama, Jun-Ichi; Morokuma, Kazunori; Ohkuma, Kunio; Goto, Norihisa; Tochikubo, Kunio

    2004-08-13

    Recombinant cholera toxin B subunit (rCTB) which is produced by Bacillus brevis carrying pNU212-CTB acts as a mucosal adjuvant capable of enhancing host immune responses specific to unrelated, mucosally co-administered vaccine antigens. When mice were administered intranasally with diphtheria-pertussis-tetanus (DPT) combination vaccine consisting of diphtheria toxoid (DTd), tetanus toxoid (TTd), pertussis toxoid (PTd), and formalin-treated filamentous hemagglutinin (fFHA), the presence of rCTB elevated constantly high values of DTd- and TTd-specific serum ELISA IgG antibody titres, and protective levels of diphtheria and tetanus toxin-neutralizing antibodies but the absence of rCTB did not. Moreover, the addition of rCTB protected all mice against tetanic symptoms and deaths. DPT combination vaccine raised high levels of serum anti-PT IgG antibody titres regardless of rCTB and protected mice from Bordetella pertussis challenge. These results suggest that co-administration of rCTB as an adjuvant is necessary for induction of diphtheria and tetanus antitoxin antibodies on the occasion of intranasal administration of DPT combination vaccine.

  11. ACTION OF DIPHTHERIA TOXIN IN THE GUINEA PIG

    PubMed Central

    Baseman, Joel B.; Pappenheimer, A. M.; Gill, D. M.; Harper, Annabel A.

    1970-01-01

    The blood clearance and distribution in the tissues of 125I after intravenous injection of small doses (1.5–5 MLD or 0.08–0.25 µg) of 125I-labeled diphtheria toxin has been followed in guinea pigs and rabbits and compared with the fate of equivalent amounts of injected 125I-labeled toxoid and bovine serum albumin. Toxoid disappeared most rapidly from the blood stream and label accumulated and was retained in liver, spleen, and especially in kidney. Both toxin and BSA behaved differently. Label was found widely distributed among all the organs except the nervous system and its rate of disappearance from the tissues paralleled its disappearance from the circulation. There was no evidence for any particular affinity of toxin for muscle tissue or for a "target" organ. Previous reports by others that toxin causes specific and selective impairment of protein synthesis in muscle tissue were not confirmed. On the contrary, both in guinea pigs and rabbits, a reduced rate of protein synthesis was observed in all tissues that had taken up the toxin label. In tissues removed from intoxicated animals of both species there was an associated reduction in aminoacyl transferase 2 content. It is concluded that the primary action of diphtheria toxin in the living animal is to effect the inactivation of aminoacyl transferase 2. The resulting inhibition in rate of protein synthesis leads to morphologic damage in all tissues reached by the toxin and ultimately to death of the animal. PMID:5511567

  12. Community-based seroepidemiology of diphtheria and tetanus in Edirne, Turkey.

    PubMed

    Tansel, Ozlem; Ekuklu, Galip; Eker, Alper; Kunduracilar, Hakan; Yuluğkural, Zerrin; Yüksel, Pelin

    2009-07-01

    The aim of this study was to evaluate the seroprevalence and correlates of diphtheria and tetanus in Edirne, Turkey. Tetanus and diphtheria antitoxin levels were determined by enzyme-linked immunosorbent assay. Among 99 participants, a diphtheria antitoxin level of >or=0.1 IU/mL was found in 97 (98%), while 2 (2%) had antitoxin levels of 0.011-0.099 IU/mL. The geometric mean titres (GMTs) in men were statistically higher. Among 295 participants, a tetanus antitoxin level of >or=0.1 IU/mL was found in 291 (98.6%), while 4 (1.4%) had antitoxin levels of 0.011-0.099 IU/mL. Participants who had completed secondary school or higher education showed higher GMT values. Additionally, participants vaccinated within the previous 5 years had higher GMT values and the percentage of participants who had completed secondary school or higher education was higher among them. GMTs decrease with increasing age and increase as the poverty index increases. The average socioeconomic status index of the participants was high for both diphtheria and tetanus seroepidemiology. In this community-based study, antitoxin levels of diphtheria and tetanus were high. However, revaccination of adults with tetanus-diphtheria toxoids at every opportunity (military service, pregnancy, post-injury prophylaxis, etc.) together with a single booster every 10 years should be considered as an immunization policy.

  13. Serological immunity to diphtheria and tetanus in healthy adults in Delhi, India.

    PubMed

    Saxena, Sonal; Jais, Manoj; Dutta, Renu; Dutta, A K

    2009-07-01

    Widespread childhood immunization with DPT (diphtheria, pertussis and tetanus) has largely eradicated diphtheria and tetanus from many countries. The reduction in the circulation of toxigenic strains has resulted in less natural boosting of adult immunity. As a result, the adult population in countries with high childhood immunization coverage have become susceptible to the disease. The duration of immunity after primary immunization to diphtheria and tetanus is limited and a reduction in immunity is common in adults. With this perspective, the present study was carried out on a random serum sample of 255 healthy individuals aged 20-50 years. The serum samples were tested for immunoglobulin G levels against diphtheria and tetanus by enzyme immuno assays. Fifty-three per cent of adults were unprotected; 22 % were seen to have only a basic protection against diphtheria; 25% were protected against both diseases; and 47% were susceptible to tetanus. The susceptibility was seen to increase with age. To avoid epidemics in the future, immunity must be improved. It is important to treat even the most trivial wound with care and tetanus toxoid immunization. Also, it is necessary to monitor the community for immunity to diphtheria using standard techniques in order to undertake epidemiological surveillances of, and prevention from, these dreadful diseases.

  14. Does vaccination ensure protection? Assessing diphtheria and tetanus antibody levels in a population of healthy children

    PubMed Central

    Gowin, Ewelina; Wysocki, Jacek; Kałużna, Ewelina; Świątek-Kościelna, Bogna; Wysocka-Leszczyńska, Joanna; Michalak, Michał; Januszkiewicz-Lewandowska, Danuta

    2016-01-01

    Abstract Vaccination effectiveness is proven when the disease does not develop after a patient is exposed to the pathogen. In the case of rare diseases, vaccination effectiveness is assessed by monitoring specific antibody levels in the population. Such recurrent analyses allow the evaluation of vaccination programs. The primary schedule of diphtheria and tetanus vaccinations is similar in various countries, with differences mainly in the number and timing of booster doses. The aim of the study was to assess diphtheria and tetanus antibody concentrations in a population of healthy children. Diphtheria and tetanus antibody levels were analyzed in a group of 324 children aged 18 to 180 months. All children were vaccinated in accordance with the Polish vaccination schedule. Specific antibody concentrations greater than 0.1 IU/mL were considered protective against tetanus or diphtheria. Levels above 1.0 were considered to ensure long-term protection. Protective levels of diphtheria antibodies were found in 229 patients (70.46%), and of tetanus in 306 patients (94.15%). Statistically significant differences were found in tetanus antibody levels in different age groups. Mean concentrations and the percentage of children with high tetanus antibody titers increased with age. No similar correlation was found for diphtheria antibodies. High diphtheria antibody levels co-occurred in 72% of the children with high tetanus antibody levels; 95% of the children with low tetanus antibody levels had low levels of diphtheria antibodies. The percentage of children with protective diphtheria antibody levels is lower than that in the case of tetanus antibodies, both in Poland and abroad, but the high proportion of children without diphtheria protection in Poland is an exception. This is all the more puzzling when taking into account that Polish children are administered a total of 5 doses containing a high concentration of diphtheria toxoid, at intervals shorter than 5 years. The

  15. Genome organization and pathogenicity of Corynebacterium diphtheriae C7(-) and PW8 strains.

    PubMed

    Iwaki, Masaaki; Komiya, Takako; Yamamoto, Akihiko; Ishiwa, Akiko; Nagata, Noriyo; Arakawa, Yoshichika; Takahashi, Motohide

    2010-09-01

    Corynebacterium diphtheriae is the causative agent of diphtheria. In 2003, the complete genomic nucleotide sequence of an isolate (NCTC13129) from a large outbreak in the former Soviet Union was published, in which the presence of 13 putative pathogenicity islands (PAIs) was demonstrated. In contrast, earlier work on diphtheria mainly employed the C7(-) strain for genetic analysis; therefore, current knowledge of the molecular genetics of the bacterium is limited to that strain. However, genomic information on the NCTC13129 strain has scarcely been compared to strain C7(-). Another important C. diphtheriae strain is Park-Williams no. 8 (PW8), which has been the only major strain used in toxoid vaccine production and for which genomic information also is not available. Here, we show by comparative genomic hybridization that at least 37 regions from the reference genome, including 11 of the 13 PAIs, are considered to be absent in the C7(-) genome. Despite this, the C7(-) strain still retained signs of pathogenicity, showing a degree of adhesion to Detroit 562 cells, as well as the formation of and persistence in abscesses in animal skin comparable to that of the NCTC13129 strain. In contrast, the PW8 strain, suggested to lack 14 genomic regions, including 3 PAIs, exhibited more reduced signs of pathogenicity. These results, together with great diversity in the presence of the 37 genomic regions among various C. diphtheriae strains shown by PCR analyses, suggest great heterogeneity of this pathogen, not only in genome organization, but also in pathogenicity.

  16. The 1925 Diphtheria Antitoxin Run to Nome - Alaska: A Public Health Illustration of Human-Animal Collaboration.

    PubMed

    Aboul-Enein, Basil H; Puddy, William C; Bowser, Jacquelyn E

    2016-12-29

    Diphtheria is an acute toxin-mediated superficial infection of the respiratory tract or skin caused by the aerobic gram-positive bacillus Corynebacterium diphtheriae. The epidemiology of infection and clinical manifestations of the disease vary in different parts of the world. Historical accounts of diphtheria epidemics have been described in many parts of the world since antiquity. Developed in the late 19th century, the diphtheria antitoxin (DAT) played a pivotal role in the history of public health and vaccinology prior to the advent of the diphtheria-tetanus toxoids and acellular pertussis (DTaP) vaccine. One of the most significant demonstrations of the importance of DAT was its use in the 1925 diphtheria epidemic of Nome, Alaska. Coordinated emergency delivery of this life-saving antitoxin by dog-sled relay in the harshest of conditions has left a profound legacy in the annals of vaccinology and public health. Lead dogs Balto and Togo, and the dog-led antitoxin run of 1925 represent a dynamic illustration of the contribution made by non-human species towards mass immunization in the history of vaccinology. This unique example of cooperative interspecies fellowship and collaboration highlights the importance of the human-animal bond in the one-health initiative.

  17. Diphtheria on Skid Road, Seattle, Wash., 1972-75.

    PubMed Central

    Pedersen, A H; Spearman, J; Tronca, E; Bader, M; Harnisch, J

    1977-01-01

    From July 1972 to December 1975, an unusual outbreak of diphtheria in Seattle, Wash., resulted in a total of 558 cases and carriers, mostly among heavy alcohol users. Skin infections were predominant. Four white men died. The highest attack rate was among native American Indians. Environmental contamination and poor personal hygience were believed to be important in continuation of the epidemic, but could not be proved. Control measures included casefinding, isolation and quarantine, sanitizing dwelling units and mass immunization with Td toxoid. The high-risk geographic area was the city's Skid Road. This area continues to be the reservoir of continuing infection, but not all population subgroups there have been at equal risk. Spread to other geographic areas of the city and county has been minimal and remains under control. PMID:877208

  18. Simultaneous quantitation of diphtheria and tetanus antibodies by double antigen, time-resolved fluorescence immunoassay.

    PubMed

    Aggerbeck, H; Nørgaard-Pedersen, B; Heron, I

    1996-04-19

    A dual, double antigen, time-resolved fluorescence immunoassay (DELFIA) for the simultaneous detection and quantitation of diphtheria (D) and tetanus (T) antibodies in sera has been developed. In the double antigen format one arm of the antibody binds to antigen coated microtitre wells and the other arm binds to labelled antigen to provide a fluorescent signal. This assay was found to be functionally specific for IgG antibodies and showed a good correlation with established toxin neutralization assays. Furthermore, the double antigen set-up was species independent, permitting the direct use of existing international references of animal origin to measure protective antibody levels in humans in international units (IU/ml). The detection limit corresponded to 0.0003 IU/ml with Eu(3+)-labelled toxoids and to 0.0035 IU/ml using Sm(3+)-labelled toxoids. The assay was fast with a high capacity making it a suitable method for serological surveillance studies.

  19. [Serum sickness in diphtheria].

    PubMed

    Vozianova, Zh I; Chepilko, K I

    1999-01-01

    As many as 2247 patients with different clinical forms of diphtheria were examined. Antidiphtheric serum (ADS) was administered in 1556 children, the dosage being determined by condition of the patient. Serum sickness developed at day 7 to 9 in 24 (1.5%); 10 patients were found to run a mild course, 14--moderately severe. 6 patients had allergic reactions: 3--to antibiotic (penicillin), urticaria type, 1--to pertussoid-tetanic anatoxin, 2 had pollinosis-type reaction. Thus, serum sickness has practical value, which fact requires a detailed allergic history together with skin tests to be performed before the administration of ADS.

  20. Adjuvants for Clostridium tetani and Clostridium diphtheriae vaccines updating.

    PubMed

    Alshanqiti, Fatimah M; Al-Masaudi, Saad B; Al-Hejin, Ahmed M; Redwan, Elrashdy M

    2017-01-01

    It's known that diphtheria and tetanus are a contagious lethal diseases over the years, they caused by pathogenic microbes corynebacterium diphtheria and Clostridium tetani, respectively. The diseases result from the production of bacterial toxin. Vaccination with bacterial toxoid vaccines adsorbed on particulates adjuvants still are the best way to prevent this epidemic diseases from spread. The particulate vaccines have been shown to be more efficient than soluble one for the induction of the immune responses. Nanoparticles can be engineered to enhance the immune responses. As well known the immune response to inactivate killed and subunit vaccine enhances by alum adjuvants. The adjuvants examined and tested after reducing its size to particle size, thus mimic size of viruses which is considered smallest units can derive the immune system. The major issue is minimizing the adjuvant particles, to gain insight of resulting immunity types and impact on immune response. The adjuvant effect of micro/nanoparticles appears to largely be a consequence of their uptake into antigen presenting cells.

  1. Collaborative study for establishment of the European Pharmacopoeia BRP batch 1 for diphtheria toxin.

    PubMed

    Sesardic, D; Prior, C; Daas, A; Buchheit, K H

    2003-07-01

    A stable liquid candidate Biological Reference Preparation (BRP) for diphtheria toxin was prepared in peptone buffer (nominal content of diphtheria toxin: 1 Lf/ml, 0.4 micro g/ml), filled in ampoules (filling volume: 1 ml) and characterised in a collaborative study. The toxin is to be used in the test "Absence of toxin and irreversibility of toxoid" as described in the current European Pharmacopoeia (Ph. Eur.) monograph Diphtheria Vaccine (Adsorbed) (2002:0443). Eleven laboratories assessed the specific activity of the preparation by in vivo and in vitro assays. The material is assumed to have satisfactory stability with a calculated predicted loss of activity of <1% per year at 4-8 degrees C. From the collaborative study, the specific activity was calculated as 77.6 (45-113) LD( 50)/ml (lethal challenge) and >75 000 Lr/Lf (intradermal challenge). The candidate BRP was successfully used in nine laboratories and confirmed suitable for use in the Vero cell test for "Absence of toxin and irreversibility of toxoid" as described in the Ph. Eur. monograph 2002:0443; i.e., concentrations of 5 x 10( -5) Lf/ml and below caused cytotoxic effects in the Vero cell test. Due to its liquid nature, the stability of the material will be monitored at regular intervals and preparation of a stable freeze-dried formulation will be considered for long-term use. Additional studies will be performed to confirm suitability of this BRP for other applications. The candidate BRP was adopted as the Ph. Eur. reference material for Diphtheria Toxin Batch 1 by the Ph. Eur. Commission at its session in March 2003.

  2. Oral immunogenicity of tomato-derived sDPT polypeptide containing Corynebacterium diphtheriae, Bordetella pertussis and Clostridium tetani exotoxin epitopes.

    PubMed

    Soria-Guerra, Ruth E; Rosales-Mendoza, Sergio; Moreno-Fierros, Leticia; López-Revilla, Rubén; Alpuche-Solís, Angel G

    2011-03-01

    DPT vaccine, designed to immunize against diphtheria, pertussis, and tetanus, has been shown to be effective in humans. Nevertheless, dissatisfaction with the whole-cell preparations is due to the reactogenicity, which has to lead to the development of new safer formulations. Previously, we described the expression in tomato of a plant-optimized synthetic gene encoding the recombinant polypeptide sDPT, containing mainly immunoprotective epitopes of the diphtheria, pertussis and tetanus exotoxins and two adjuvants. In this study, we examined whether the ingestion of tomato-derived sDPT protein induces specific antibodies in mice after three weekly doses scheme. A positive group immunized with DPT toxoids was included. Specific antibody levels were assessed in serum, gut and lung. Sera tested for IgG antibody response to pertussis, tetanus and diphtheria toxin showed responses to the foreign antigens; interestingly, the response to diphtheria epitope was similar to those observed in the positive group. We found higher IgG1 than IgG2a responses in serum. A modest IgG response was observed in the tracheopulmonary fluid. High response of IgA against tetanus toxin was evident in gut, which was statistically comparable to that obtained in the positive group. The levels of response in these groups were higher than those in mice that received wild-type tomato. These findings support the concept of using transgenic tomatoes expressing sDPT polypeptide as model for edible vaccine against diphtheria, pertussis, and tetanus.

  3. Prevention of pertussis, tetanus, and diphtheria among pregnant and postpartum women and their infants recommendations of the Advisory Committee on Immunization Practices (ACIP).

    PubMed

    Murphy, Trudy V; Slade, Barbara A; Broder, Karen R; Kretsinger, Katrina; Tiwari, Tejpratap; Joyce, Patricia M; Iskander, John K; Brown, Kristin; Moran, John S

    2008-05-30

    In 2005, two tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (Tdap) vaccines were licensed and recommended for use in adults and adolescents in the United States: ADACEL (sanofi pasteur, Swiftwater, Pennsylvania), which is licensed for use in persons aged 11--64 years, and BOOSTRIX (GlaxoSmithKline Biologicals, Rixensart, Belgium), which is licensed for use in persons aged 10-18 years. Both Tdap vaccines are licensed for single-dose use to add protection against pertussis and to replace the next dose of tetanus and diphtheria toxoids vaccine (Td). Available evidence does not address the safety of Tdap for pregnant women, their fetuses, or pregnancy outcomes sufficiently. Available data also do not indicate whether Tdap-induced transplacental maternal antibodies provide early protection against pertussis to infants or interfere with an infant's immune responses to routinely administered pediatric vaccines. Until additional information is available, CDC's Advisory Committee on Immunization Practices recommends that pregnant women who were not vaccinated previously with Tdap: 1) receive Tdap in the immediate postpartum period before discharge from hospital or birthing center, 2) may receive Tdap at an interval as short as 2 years since the most recent Td vaccine, 3) receive Td during pregnancy for tetanus and diphtheria protection when indicated, or 4) defer the Td vaccine indicated during pregnancy to substitute Tdap vaccine in the immediate postpartum period if the woman is likely to have sufficient protection against tetanus and diphtheria. Although pregnancy is not a contraindication for receiving Tdap vaccine, health-care providers should weigh the theoretical risks and benefits before choosing to administer Tdap vaccine to a pregnant woman. This report 1) describes the clinical features of pertussis, tetanus, and diphtheria among pregnant and postpartum women and their infants, 2) reviews available evidence of pertussis vaccination during

  4. Divalent toxoids loaded stable chitosan-glucomannan nanoassemblies for efficient systemic, mucosal and cellular immunostimulatory response following oral administration.

    PubMed

    Harde, Harshad; Siddhapura, Krupa; Agrawal, Ashish Kumar; Jain, Sanyog

    2015-06-20

    The present study reports dual tetanus and diphtheria toxoids loaded stable chitosan-glucomannan nanoassemblies (sCh-GM-NAs) formulated using tandem ionic gelation technique for oral mucosal immunization. The stable, lyophilized sCh-GM-NAs exhibited ~152 nm particle size and ~85% EE of both the toxoids. The lyophilized sCh-GM-NAs displayed excellent stability in biomimetic media and preserved chemical, conformation and biological stability of encapsulated toxoids. The higher intracellular APCs uptake of sCh-GM-NAs was concentration and time dependent which may be attributed to the receptor mediated endocytosis via mannose and glucose receptor. The higher Caco-2 uptake of sCh-GM-NAs was further confirmed by ex vivo intestinal uptake studies. The in vivo evaluation revealed that sCh-GM-NAs posed significantly (p<0.001) higher humoral, mucosal and cellular immune response than other counterparts by eliciting complete protective levels of anti-TT and anti-DT (~0.1 IU/mL) antibodies. Importantly, commercial 'Dual antigen' vaccine administered through oral or intramuscular route was unable to elicit all type of immune response. Conclusively, sCh-GM-NAs could be considered as promising vaccine adjuvant for oral mucosal immunization.

  5. Does vaccination ensure protection? Assessing diphtheria and tetanus antibody levels in a population of healthy children: A cross-sectional study.

    PubMed

    Gowin, Ewelina; Wysocki, Jacek; Kałużna, Ewelina; Świątek-Kościelna, Bogna; Wysocka-Leszczyńska, Joanna; Michalak, Michał; Januszkiewicz-Lewandowska, Danuta

    2016-12-01

    Vaccination effectiveness is proven when the disease does not develop after a patient is exposed to the pathogen. In the case of rare diseases, vaccination effectiveness is assessed by monitoring specific antibody levels in the population. Such recurrent analyses allow the evaluation of vaccination programs. The primary schedule of diphtheria and tetanus vaccinations is similar in various countries, with differences mainly in the number and timing of booster doses. The aim of the study was to assess diphtheria and tetanus antibody concentrations in a population of healthy children.Diphtheria and tetanus antibody levels were analyzed in a group of 324 children aged 18 to 180 months. All children were vaccinated in accordance with the Polish vaccination schedule.Specific antibody concentrations greater than 0.1 IU/mL were considered protective against tetanus or diphtheria. Levels above 1.0 were considered to ensure long-term protection.Protective levels of diphtheria antibodies were found in 229 patients (70.46%), and of tetanus in 306 patients (94.15%). Statistically significant differences were found in tetanus antibody levels in different age groups. Mean concentrations and the percentage of children with high tetanus antibody titers increased with age. No similar correlation was found for diphtheria antibodies. High diphtheria antibody levels co-occurred in 72% of the children with high tetanus antibody levels; 95% of the children with low tetanus antibody levels had low levels of diphtheria antibodies.The percentage of children with protective diphtheria antibody levels is lower than that in the case of tetanus antibodies, both in Poland and abroad, but the high proportion of children without diphtheria protection in Poland is an exception. This is all the more puzzling when taking into account that Polish children are administered a total of 5 doses containing a high concentration of diphtheria toxoid, at intervals shorter than 5 years. The decrease in

  6. Quantitation of anti-tetanus and anti-diphtheria antibodies by enzymoimmunoassay: methodology and applications.

    PubMed

    Virella, G; Hyman, B

    1991-01-01

    We have developed enzymoimmunoassays (EIA) for the quantitation of antibodies (Ab) to tetanus and diphtheria toxoids (TT, DT) using Immulon I plates coated with the appropriate toxoid. A preparation of human tetanus immunoglobulin with a known concentration of anti-TT Ab was used as calibrator of the anti-TT antibody assay. The assay of anti-DT Ab is calibrated with a pool of human sera whose anti-DT Ab concentration was determined by quantitative immunoelectrophoresis, using a horse anti-DT with known Ab concentration as calibrator. A peroxidase-conjugated anti-human IgG was used in both assays. ABTS was used as substrate, and the reaction was stopped after 1 min incubation with citric acid and the OD measured at 414 nm on a Vmax reader. The assays have been applied to a variety of clinical situations. In patients suspected of having tetanus, the quantitation of antibodies has been helpful in establishing a diagnosis. In patients with a history of hypersensitivity to tetanus toxoid, verification of the levels of anti-TT antibody may prevent unnecessary and potentially harmful immunizations. The assays have also been used for the diagnostic evaluation of the humoral immune response to TT and DT, both in pediatric patients and in immunosuppressed patients. Several non-responders have been detected, and we have recently used the assay to monitor the effects of fish oil administration on the humoral immune response.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test.

    PubMed

    Sonobe, M H; Trezena, A G; Guilhen, F B; Takano, V L; Fratelli, F; Sakauchi, D; Morais, J F; Prado, S M A; Higashi, H G

    2007-01-01

    A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r(2) = 0.95, P < 0.0001) and for diphtheria (r(2) = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

  8. Biochemical and biological characteristics of cross-reacting material 197 CRM197, a non-toxic mutant of diphtheria toxin: use as a conjugation protein in vaccines and other potential clinical applications.

    PubMed

    Bröker, Michael; Costantino, Paolo; DeTora, Lisa; McIntosh, E David; Rappuoli, Rino

    2011-07-01

    The biochemical and biological characteristics of CRM(197) are reviewed. Polysaccharide protein conjugate vaccines represent an important technological advancement that allowed for protection against dangerous diseases in vulnerable populations such as infants. The first carrier proteins, diphtheria and tetanus toxoids, were chosen in the context of an extensive body of information describing their immunogenicity and safety profiles in clinical use. These carriers perform well, and they require detoxification. A non-toxic mutant of diphtheria toxin, cross-reacting material 197 (CRM(197)), is a useful carrier protein with several manufacturing and other potential advantages over toxoids. For over a decade, several important and widely used routine childhood glycoconjugate vaccines against serious illnesses, including Haemophilus influenzae type b and pneumococcal disease, have employed CRM(197) as carrier protein. Additional clinical applications of CRM(197), as in chemotherapy, also exist.

  9. Safety and immunogenicity of a combined Tetanus, Diphtheria, recombinant acellular Pertussis vaccine (TdaP) in healthy Thai adults

    PubMed Central

    Sirivichayakul, Chukiat; Chanthavanich, Pornthep; Limkittikul, Kriengsak; Siegrist, Claire-Anne; Wijagkanalan, Wassana; Chinwangso, Pailinrut; Petre, Jean; Hong Thai, Pham; Chauhan, Mukesh; Viviani, Simonetta

    2017-01-01

    ABSTRACT Background: An acellular Pertussis (aP) vaccine containing recombinant genetically detoxified Pertussis Toxin (PTgen), Filamentous Hemagglutinin (FHA) and Pertactin (PRN) has been developed by BioNet-Asia (BioNet). We present here the results of the first clinical study of this recombinant aP vaccine formulated alone or in combination with tetanus and diphtheria toxoids (TdaP). Methods: A phase I/II, observer-blind, randomized controlled trial was conducted at Mahidol University in Bangkok, Thailand in healthy adult volunteers aged 18–35 y. The eligible volunteers were randomized to receive one dose of either BioNet's aP or Tetanus toxoid-reduced Diphtheria toxoid-acellular Pertussis (TdaP) vaccine, or the Tdap Adacel® vaccine in a 1:1:1 ratio. Safety follow-up was performed for one month. Immunogenicity was assessed at baseline, at 7 and 28 d after vaccination. Anti-PT, anti-FHA, anti-PRN, anti-tetanus and anti-diphtheria IgG antibodies were assessed by ELISA. Anti-PT neutralizing antibodies were assessed also by CHO cell assay. Results: A total of 60 subjects (20 per each vaccine group) were enrolled and included in the safety analysis. Safety laboratory parameters, incidence of local and systemic post-immunization reactions during 7 d after vaccination and incidence of adverse events during one month after vaccination were similar in the 3 vaccine groups. One month after vaccination, seroresponse rates of anti-PT, anti-FHA and anti-PRN IgG antibodies exceeded 78% in all vaccine groups. The anti-PT IgG, anti-FHA IgG, and anti-PT neutralizing antibody geometric mean titers (GMTs) were significantly higher following immunization with BioNet's aP and BioNet's TdaP than Adacel® (P< 0.05). The anti-PRN IgG, anti-tetanus and anti-diphtheria GMTs at one month after immunization were comparable in all vaccine groups. All subjects had seroprotective titers of anti-tetanus and anti-diphtheria antibodies at baseline. Conclusion: In this first clinical study

  10. [The local manifestations of diphtheria].

    PubMed

    Kapustian, V A; Boldyrev, V V; Maleev, V V; Mikhaĭlova, E I; Sedak, E F

    1994-01-01

    The development of diphtheria is characterized by a pronounced local process, but the description of local changes in the disease are based only on postmortem findings. 67 patients with different forms of diphtheria were examined. In 11 cases of descending croup bronchoscopic examination was carried out. As revealed in this study, at the primary stage of the disease films cover the whole of the air duct system, and the process is identical to that on tonsils. In the course of convalescence and under the action of specific antiserum films on the tonsils, the soft palate and the vocal cords disappeared, and films in the larynx, the trachea and bronchi could be easily separated from the mucous membrane, but always with the formation of erosions and even perforations in it. The character of the process was indicative of the absence of any specific action of diphtheria toxin on the mucous membrane at the site of the inoculation of the infective agent. No correlation between the severity of the course of diphtheria, the degree of edema and the frequency of the development of complications was noted.

  11. Anaphylactic reaction to diphtheria-tetanus vaccine in a child: specific IgE/IgG determinations and cross-reactivity studies.

    PubMed

    Martín-Muñoz, M Flora; Pereira, M José; Posadas, Sinforiano; Sánchez-Sabaté, Elena; Blanca, Miguel; Alvarez, Javier

    2002-09-10

    The present study describes the occurrence of an anaphylactic reaction after the administration of the fifth booster dose of DT vaccine in a six-year-old child. Skin test, in vitro determinations of specific IgE antibodies and immunoblotting assays showed that the IgE response was directed against tetanus and diphtheria toxoids (Dtx). IgG antibodies were also detected by ELISA and immunoblotting. The RAST and immunoblotting inhibitions showed no cross-reactivity between the two toxoids, indicating the presence of co-existing but non-cross-reacting IgE and IgG antibodies. This was maintained in two subsequent determinations done 18 and 30 months after the episode. To our knowledge, this is the first study of cross-reactivity between tetanus and diphtheria antigens. We show that simultaneous IgE antibodies to two different toxoids may occur, indicating that after an immediate reaction to DT, a search for IgE antibodies to both tetanus and Dtx should be undertaken.

  12. Clinical study of transcutaneous vaccination using a hydrogel patch for tetanus and diphtheria.

    PubMed

    Hirobe, Sachiko; Matsuo, Kazuhiko; Quan, Ying-Shu; Kamiyama, Fumio; Morito, Hironori; Asada, Hideo; Takaya, Yusuke; Mukai, Yohei; Okada, Naoki; Nakagawa, Shinsaku

    2012-02-27

    Transcutaneous immunization (TCI) is a non-invasive and easy-to-use vaccination method. We demonstrated the efficacy and safety of a transcutaneous vaccine formulation using a hydrogel patch in animal experiments. In the present study, we performed a clinical study to apply our TCI formulation for vaccination against tetanus and diphtheria in human. The TCI device was a hydrogel patch (antigen-free) applied to the left brachial medial skin of 22 healthy volunteers for 48 h. Next, the hydrogel patch, containing 2mg tetanus toxoid (TT) and 2mg diphtheria toxoid (DT) as the TCI formulation, was applied to 27 healthy volunteers for 24h and some volunteers were vaccinated again by TCI formulation. For safety assessment, the patch application site was observed to assess local adverse events, and systemic adverse events were determined by a blood test. The antigen-free hydrogel patch and TCI formulation containing TT and DT did not induce local or systemic severe adverse events. For vaccine efficacy estimation, toxoid-specific serum antibody titers were determined by ELISA and the toxin-neutralizing activity of the induced antibody was evaluated in a passive-challenge experiment. The anti-TT IgG titer and the anti-DT IgG titer increased, and a significant effect was detected by paired t-test. The antibody titers were maintained at higher level than that before vaccination for at least 1 year. Moreover, toxoid-specific antibodies were produced by the second vaccination in some subjects. Antibodies induced by application of the TCI formulation neutralized the toxin and prevented toxic death in mice. In addition, changes in the skin condition due to application of the TCI formulation were observed under in vivo confocal Raman spectroscopy. The amount of water and patch components in the stratum corneum increased after application of the TCI formulation, suggesting that the change in the skin condition was related to antigen penetration. These data indicate that this easy

  13. Potency tests of diphtheria, tetanus and combined vaccines. Suggestion for a simplified potency assay.

    PubMed

    Aggerbeck, H; Heron, I

    1996-01-01

    Two diphtheria-tetanus vaccines (DT), adsorbed to either aluminium hydroxide or calcium phosphate but identical with respect to toxoid origin and amounts, were compared in full potency assays in mice according to the European Pharmacopoeia (EP) and in a reduced potency assay in guinea-pigs using a double dose immunization schedule. The efficacy of the vaccines was compared in a clinical trial with revaccination of 313 military recruits. The reduced potency assay gave a better reflection of the efficacy of the two vaccines in humans than the required assays of the EP. For release of combined, final vaccine formulations the reduced potency assay suggested will reduce the number of animals in quality control.

  14. An in vitro immune response model to determine tetanus toxoid antigen (vaccine) specific immunogenicity: Selection of sensitive assay criteria.

    PubMed

    Piersma, Sytse J; Leenaars, Marlies P P A M; Guzylack-Piriou, Laurence; Summerfield, Artur; Hendriksen, Coenraad F M; McCullough, Ken C

    2006-04-12

    Many vaccines employed in childhood vaccination programmes are produced by conventional techniques, resulting in complex biological mixtures for which batch-related quality control requires in vivo potency testing. Monitoring consistency via in vitro tests during the vaccine production has the capacity to replace certain of the in vivo methods. In this respect, determining vaccine antigen immunogenicity through functional immunological tests has high potential. Advances in immunology have made it possible to analyse this biological activity by in vitro means. The present study established such an in vitro test system for tetanus toxoid (TT). This measured vaccine immunogenicity through an antigen-specific secondary (recall) response in vitro, using a porcine model growing in value for its closeness to human immune response characteristics. Discrimination between the specific recall TT antigen and diphtheria toxoid (DT) was possible using both peripheral blood mononuclear cell cultures and monocyte-derived dendritic cells in co-culture with autologous specific lymphocytes. TT-specific activation was detected with highest discrimination capacity using proliferation assays, as well as IFN-gamma and TT-specific antibody ELISPOTS (measuring secreting T and B lymphocytes, respectively). These in vitro systems show a high potential for replacing animal experimentation to evaluate the immunogenicity of complex vaccines.

  15. Booster vaccination against diphtheria and tetanus in man. Comparison of three different vaccine formulations--III.

    PubMed

    Aggerbeck, H; Wantzin, J; Heron, I

    1996-09-01

    Adverse reactions and antibody levels were compared following a booster vaccination of 177 Danish military recruits with a plain, an aluminium hydroxide (0.5 mg Al per human dose, HD) and a calcium phosphate (0.25 mg Ca per HD) adsorbed diphtheria-tetanus (D-T) vaccine. The calcium phosphate adsorbed vaccine was given in a HD of 3 Lf of D and T toxoids and proved to be of equal efficacy as the aluminium hydroxide adsorbed vaccine which was injected in a dose containing twice the antigen amount. The calcium phosphate vaccine caused fewer adverse reactions than the one adsorbed to aluminium hydroxide. The plain vaccine (6 Lf per HD of D and T toxoid) had the highest efficacy with a similar low occurrence of adverse reactions as the calcium phosphate adsorbed vaccine. Potency assays in mice were in accordance with these immunogenicity results in man if a two dose immunization schedule was followed, but not if the vaccines were compared after a single immunization as requested by the procedure for potency testing according to current WHO and European Pharmacopoeia requirements. Both of the adsorbed vaccines primed mice for specific IgE antibody formation. This could be detected after a second immunization with either of the adsorbed vaccines or with the plain D-T vaccine. Also in humans, immunization with the plain vaccine boosted specific IgE formation to a detectable level. This may be ascribed to adjuvant priming during the primary vaccination series some 20 years previously.

  16. Epidemic diphtheria in the Republic of Georgia, 1993-1996: risk factors for fatal outcome among hospitalized patients.

    PubMed

    Quick, M L; Sutter, R W; Kobaidze, K; Malakmadze, N; Strebel, P M; Nakashidze, R; Murvanidze, S

    2000-02-01

    Epidemic diphtheria reemerged in the republic of Georgia in November 1993. To identify risk factors for fatal outcomes, clinical and epidemiologic data on all hospitalized diphtheria patients were examined. Medical charts of patients from 1993-1995 were reviewed. A total of 659 cases and 68 deaths were identified (case fatality rate [CFR] = 10.3%). Fifty-two percent of all cases and 68% of deaths were in children diphtheria toxoid and adults 40-49 years of age were the 2 groups at highest risk. Being a rural resident and having a long interval (>3 days) between onset of symptoms to antitoxin treatment were significantly associated with fatal outcomes. Immunization of children and 40- to 49-year-old adults was required to rapidly control the epidemic.

  17. Seroepidemiology of diphtheria and pertussis in Beijing, China: A cross-sectional study.

    PubMed

    Li, Xiaomei; Chen, Meng; Zhang, Tiegang; Li, Juan; Zeng, Yang; Lu, Li

    2015-01-01

    The aim of this study was to assess the level of humoral immunity against diphtheria and pertussis by measuring IgG to diphtheria toxoid (DT) and pertussis toxin (PT) in general population of Beijing. A total of 2147 subjects aged 0-74 y were selected with a random sample of resident population in Beijing. The information of socio-demographic characteristics, vaccination history, disease history of diphtheria and pertussis were collected for each subject by questionnaire. Serum samples were tested for IgG antibodies to DT and PT by using commercial ELISA kits. The overall positivity rate of anti-DT IgG was 66.28% with the mean concentration of 2.169 IU/ml. Age stratified data showed that the highest positivity rate of 97.63% was observed in 1-4 y and the rates decreased with age. The positivity rates were only around 50% or below since 25 y old. The positivity rate of anti-PT IgG was 12.34% with the mean concentration of 15.163 IU/ml. The highest level of positivity rate (22.23%) and antibody level (23.101 IU/ml) was seen in <1 year old. In subjects older than 10 y old, the anti-PT IgG positivity rate was 10.19%-13.51% and concentration was 13.295 IU/ml -16.353 IU/ml, with no significant differences between these groups (χ2 = 1.664, P = 0.948; F = 0.369, P = 0.899). The subjects with anti-PT IgG ≥ 100 IU/ml were observed in nearly all the groups older than 5 y except for 10-14 age group. The estimated incidences of pertussis infection were higher than 6000/100000 in these age groups. A sharp increase of immunity level of diphtheria was observed at 1 y and 6 y respectively, which was consistent with the current immunization schedule. But there was no significant increase of immunity to pertussis observed after booster immunization at 18-24 months, but the proportions of undetectable were lowest in <1, 1, 2 years in children <14 years. As shown in the present study, the adult population was generally lack of protective antibody against diphtheria and all the age

  18. Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients.

    PubMed

    Mitchell, Duane A; Batich, Kristen A; Gunn, Michael D; Huang, Min-Nung; Sanchez-Perez, Luis; Nair, Smita K; Congdon, Kendra L; Reap, Elizabeth A; Archer, Gary E; Desjardins, Annick; Friedman, Allan H; Friedman, Henry S; Herndon, James E; Coan, April; McLendon, Roger E; Reardon, David A; Vredenburgh, James J; Bigner, Darell D; Sampson, John H

    2015-03-19

    After stimulation, dendritic cells (DCs) mature and migrate to draining lymph nodes to induce immune responses. As such, autologous DCs generated ex vivo have been pulsed with tumour antigens and injected back into patients as immunotherapy. While DC vaccines have shown limited promise in the treatment of patients with advanced cancers including glioblastoma, the factors dictating DC vaccine efficacy remain poorly understood. Here we show that pre-conditioning the vaccine site with a potent recall antigen such as tetanus/diphtheria (Td) toxoid can significantly improve the lymph node homing and efficacy of tumour-antigen-specific DCs. To assess the effect of vaccine site pre-conditioning in humans, we randomized patients with glioblastoma to pre-conditioning with either mature DCs or Td unilaterally before bilateral vaccination with DCs pulsed with Cytomegalovirus phosphoprotein 65 (pp65) RNA. We and other laboratories have shown that pp65 is expressed in more than 90% of glioblastoma specimens but not in surrounding normal brain, providing an unparalleled opportunity to subvert this viral protein as a tumour-specific target. Patients given Td had enhanced DC migration bilaterally and significantly improved survival. In mice, Td pre-conditioning also enhanced bilateral DC migration and suppressed tumour growth in a manner dependent on the chemokine CCL3. Our clinical studies and corroborating investigations in mice suggest that pre-conditioning with a potent recall antigen may represent a viable strategy to improve anti-tumour immunotherapy.

  19. Diphtheria: a zoonotic disease in France?

    PubMed

    Bonmarin, Isabelle; Guiso, Nicole; Le Flèche-Matéos, Anne; Patey, Olivier; Patrick, A D Grimont; Levy-Bruhl, Daniel

    2009-06-24

    Thanks to vaccination, diphtheria has almost disappeared in France. The case definition, used for mandatory notification, was expanded in 2003 to include toxin-producing strains of Corynebacterium ulcerans. We describe the epidemiology of diphtheria in France from 1990 to 2008. No cases occurred between 1990 and 2001. Since 2002, 19 cases have been reported: 4 cases due to Corynebacterium diphtheriae related to exposure in endemic countries, and 15 cases due to other corynebacteria, including 4 cases of pseudomembranous pharyngitis, mainly related to contact with domestic animals. High vaccination coverage in the population and sensitive surveillance need to be maintained. Moreover, control measures need to be adapted to the non-C. diphtheriae toxigenic species.

  20. Draft genome sequence of Corynebacterium diphtheriae biovar intermedius NCTC 5011.

    PubMed

    Sangal, Vartul; Tucker, Nicholas P; Burkovski, Andreas; Hoskisson, Paul A

    2012-09-01

    We report an annotated draft genome of the human pathogen Corynebacterium diphtheriae bv. intermedius NCTC 5011. This strain is the first C. diphtheriae bv. intermedius strain to be sequenced, and our results provide a useful comparison to the other primary disease-causing biovars, C. diphtheriae bv. gravis and C. diphtheriae bv. mitis. The sequence has been deposited at DDBJ/EMBL/GenBank with the accession number AJVH01000000.

  1. Crystallization and preliminary X-ray studies of the diphtheria Tox repressor from Corynebacterium diphtheriae.

    PubMed

    Schiering, N; Tao, X; Murphy, J R; Petsko, G A; Ringe, D

    1994-12-16

    Crystals of the diphtheria tox repressor (DtxR) from Corynebacterium diphtheriae suitable for structure determination have been obtained. DtxR activated with transition metal ions represses the expression of the structural gene for the diphtheria toxin, tox, which is encoded on the genome of a family of closely related corynebacteriophages. The space group of the obtained crystals is trigonal P3(1)21 or its enantiomorph P3(2)21 with a = b = 64.2 A, c = 220.5 A, alpha = beta = 90 degrees, gamma = 120 degrees. Two monomers comprise the asymmetric unit. The crystals diffract to a resolution of better than 3 A.

  2. Cutaneous infections due to Corynebacterium diphtheriae

    PubMed Central

    Cockcroft, W. H.; Boyko, W. J.; Allen, D. E.

    1973-01-01

    Toxigenic Corynebacterium diphtheriae was grown from skin lesions of 44 indigent patients seen at the emergency or out-patient departments of this hospital, 43 of them within the last 16 months of the study period. In all cases staphylococci or hemolytic streptococci were also present in the wounds. An increase in the incidence of clinical diphtheria occurred in the few months preceding and overlapping the period of recognition of the cutaneous infections. The gravis strains, which accounted for the majority of the infections, were sensitive to erythromycin and to penicillin, but were relatively resistant to cloxacillin. PMID:4632361

  3. Adsorption of Toll-Like Receptor 4 Agonist to Alum-Based Tetanus Toxoid Vaccine Dampens Pro-T Helper 2 Activities and Enhances Antibody Responses.

    PubMed

    Bortolatto, Juliana; Mirotti, Luciana; Rodriguez, Dunia; Gomes, Eliane; Russo, Momtchilo

    2015-01-01

    Aluminum salts gels (alum) are TLR-independent adjuvants and have been used to boost antibody responses in alum-based vaccines such as diphtheria, pertussis, and tetanus toxoid (DPT) triple vaccine. However, the pro-Th2 activity of alum-based vaccine formulations has not been fully appreciated. Here we found that alum-based tetanus toxoid (TT) vaccine was biased toward a Th-2 profile as shown by TT-induced airway eosinophilic inflammation, type 2 cytokine production, and high levels of IgE anaphylactic antibodies. The adsorption into alum of prototypic TLR4 agonists such as lipopolysaccharides (LPS) derived from Escherichia coli consistently dampened TT-induced Th2 activities without inducing IFNγ or Th1-like responses in the lung. Conversely, adsorption of monophosphoryl lipid A (MPLA) extracted from Salmonella minnesota, which is a TIR-domain-containing adapter-inducing interferon-β- (TRIF-) biased TLR4 agonist, was less effective in decreasing Th-2 responses. Importantly, in a situation with antigenic competition (OVA plus TT), TT-specific IgG1 or IgG2a was decreased compared with TT sensitization. Notably, LPS increased the production of IgG1 and IgG2a TT-specific antibodies. In conclusion, the addition of LPS induces a more robust IgG1 and IgG2a TT-specific antibody production and concomitantly decreases Th2-cellular and humoral responses, indicating a potential use of alum/TLR-based vaccines.

  4. Persistence of Corynebacterium diphtheriae in Delhi & National Capital Region (NCR).

    PubMed

    Bhagat, S; Grover, S S; Gupta, N; Roy, R D; Khare, S

    2015-10-01

    Despite the introduction of mass immunization, diphtheria continues to play a major role as a potentially lethal infectious disease in many countries. Delay in the specific therapy of diphtheria may result in death and, therefore, accurate diagnosis of diphtheria is imperative. This study was carried out at National Centre for Disease Control (NCDC), Delhi, India, on samples of suspected diphtheria cases referred from various government hospitals of Delhi and neighbouring areas during 2012-2014. Primary identification of Corynebacterium diphtheriae was done by standard culture, staining and biochemical tests followed by toxigenicity testing by Elek's test on samples positive for C. diphtheriae. The results showed persistence of toxigenic C. diphtheriae in our community indicating the possibility of inadequate immunization coverage.

  5. Targeted diphtheria toxin to treat BPDCN.

    PubMed

    FitzGerald, David J

    2014-07-17

    In this issue of Blood, Frankel et al describe a novel treatment of blastic plasmacytoid dendritic cell neoplasm (BPDCN) using an engineered version of diphtheria toxin that is targeted to malignant cells via a fusion with interleukin (IL)3 (see panel A).

  6. 9 CFR 113.115 - Staphylococcus Aureus Bacterin-Toxoid.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Staphylococcus Aureus Bacterin-Toxoid. 113.115 Section 113.115 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE..., each weighing 2000-3000 grams, shall be used as test animals. Either a five rabbit individual...

  7. 9 CFR 113.115 - Staphylococcus Aureus Bacterin-Toxoid.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Staphylococcus Aureus Bacterin-Toxoid. 113.115 Section 113.115 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE..., each weighing 2000-3000 grams, shall be used as test animals. Either a five rabbit individual...

  8. 9 CFR 113.115 - Staphylococcus Aureus Bacterin-Toxoid.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Staphylococcus Aureus Bacterin-Toxoid. 113.115 Section 113.115 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE..., each weighing 2000-3000 grams, shall be used as test animals. Either a five rabbit individual...

  9. First Draft Genome Sequences of Malaysian Clinical Isolates of Corynebacterium diphtheriae

    PubMed Central

    Ahmad, Norazah; Mohd Khalid, Mohd Khairul Nizam; Abd Wahab, Muhammad Adib; Hashim, Rohaidah; Tang, Soo Nee; Liow, Yii Ling; Hamzah, Hazwani; Dahalan, Nurul Ain; Seradja, Valentinus

    2017-01-01

    ABSTRACT Corynebacterium diphtheriae has caused multiple isolated diphtheria cases in Malaysia over the years. Here, we report the first draft genome sequences of 15 Malaysia C. diphtheriae clinical isolates collected from the years 1981 to 2016. PMID:28254972

  10. First Draft Genome Sequences of Malaysian Clinical Isolates of Corynebacterium diphtheriae.

    PubMed

    Ahmad, Norazah; Hii, Shirley Yi Fen; Mohd Khalid, Mohd Khairul Nizam; Abd Wahab, Muhammad Adib; Hashim, Rohaidah; Tang, Soo Nee; Liow, Yii Ling; Hamzah, Hazwani; Dahalan, Nurul Ain; Seradja, Valentinus

    2017-03-02

    Corynebacterium diphtheriae has caused multiple isolated diphtheria cases in Malaysia over the years. Here, we report the first draft genome sequences of 15 Malaysia C. diphtheriae clinical isolates collected from the years 1981 to 2016.

  11. Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation.

    PubMed

    Stojićević, Ivana; Dimitrijević, Ljiljana; Dovezenski, Nebojša; Živković, Irena; Petrušić, Vladimir; Marinković, Emilija; Inić-Kanada, Aleksandra; Stojanović, Marijana

    2011-08-01

    Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor.

  12. Human clinical isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans collected in Canada from 1999 to 2003 but not fitting reporting criteria for cases of diphtheria.

    PubMed

    Dewinter, Leanne M; Bernard, Kathryn A; Romney, Marc G

    2005-07-01

    A 5-year collection of Corynebacterium diphtheriae and Corynebacterium ulcerans human clinical isolates yielded nine isolates from blood cultures of patients with invasive infections, stressing the importance of C. diphtheriae as a serious blood-borne pathogen. Seven percent of C. diphtheriae and 100% of C. ulcerans isolates produced diphtheria toxin, demonstrating that toxigenic corynebacteria continue to circulate.

  13. Corynebacterium diphtheriae infections currently and in the past.

    PubMed

    Zasada, Aleksandra Anna

    2015-01-01

    Along with the introduction of common obligatory vaccinations against diphtheria, the disease has been limited in developed countries. However, diphtheria is still endemic in developing countries. Due to a growing popularity of visiting these countries, there is a risk of importation of the disease to Europe. Studies revealed that over 60% of persons aged >40 years in the Polish population do not have a protective level of antibodies against diphtheria. Furthermore, an access to diphtheria antitoxin, which is essential in diphtheria treatment, is now hardly accessible in Europe. On the other hand, in many countries, including Poland, new infections caused by non-toxigenic Corynebacterium diphtheriae have been emerged. Such infections are frequently manifested by bacteraemia and endocarditis with a high fatality rate, amounting even to 41%.

  14. Diphtheria: epidemiological update and review of prevention and control strategies.

    PubMed

    Prospero, E; Raffo, M; Bagnoli, M; Appignanesi, R; D'Errico, M M

    1997-07-01

    The importance of anti-diphtheria immunity in adults through periodic booster doses of vaccine is now increasing after last years diphtheria outbreaks in Newly Independent States (NIS) and Algeria and a few cases found in Europe and USA. Diphtheria cases notified in Italy between 1991-1994 have been reported. In 1995 WHO outlined the need to review vaccination schedules against diphtheria in all countries where gaps occur in the immunity of adults. The main sero-epidemiological studies performed in adults and vaccination schedules against diphtheria in some industrialized countries have been examined. Actual situation and control strategies adopted by WHO in the NIS and implications for other countries have been briefly presented. Finally, guidelines for management, investigation and control of diphtheria have been reported, including CDCs recommendations.

  15. New diphtheria toxin repressor types depicted in a Romanian collection of Corynebacterium diphtheriae isolates.

    PubMed

    Dinu, Sorin; Damian, Maria; Badell, Edgar; Dragomirescu, Cristiana Cerasella; Guiso, Nicole

    2014-10-01

    Corynebacterium diphtheriae is the etiological agent of diphtheria, a potential fatal disease caused by a corynephage toxin. The expression of this diphtheria toxin is controlled via an iron-dependent repressor with various functions (DtxR). Some mutations in the dtxR gene are associated with diminished activity or even with total loss of DtxR function. We conducted a molecular study to characterize the dtxR alleles harbored by 34 isolates of C. diphtheriae recovered from Romanian patients between 1961 and 2007. Three of the seven alleles identified in this study have not previously been described. Two new DtxR types were identified, one of which has an unusual polypeptide length. All the new DtxR types were found in toxigenic isolates, suggesting that they effectively regulate the expression of diphtheria toxin. Furthermore, one of the new DtxR identified was also found in a non-toxigenic isolate, making it a potential source of toxigenic isolates after lysogenic conversion.

  16. A Case of Necrotizing Epiglottitis Due to Nontoxigenic Corynebacterium diphtheriae.

    PubMed

    Lake, Jessica A; Ehrhardt, Matthew J; Suchi, Mariko; Chun, Robert H; Willoughby, Rodney E

    2015-07-01

    Diphtheria is a rare cause of infection in highly vaccinated populations and may not be recognized by modern clinicians. Infections by nontoxigenic Corynebacterium diphtheriae are emerging. We report the first case of necrotizing epiglottitis secondary to nontoxigenic C diphtheriae. A fully vaccinated child developed fever, poor oral intake, and sore throat and was found to have necrotizing epiglottitis. Necrotizing epiglottitis predominantly occurs in the immunocompromised host. Laboratory evaluation revealed pancytopenia, and bone marrow biopsy was diagnostic for acute lymphoblastic leukemia. Clinicians should be aware of aggressive infections that identify immunocompromised patients. This case highlights the features of a reemerging pathogen, C diphtheriae.

  17. [First confirmed case of laryngeal diphtheria in Djibouti].

    PubMed

    Koeck, J L; Merle, C; Bimet, F; Kiredjian, M; Goullin, B; Teyssou, R

    2000-01-01

    The first bacteriologically confirmed case of laryngeal diphtheria in Djibouti was reported in 1998. It involved a three-year-old native-born infant who had been vaccinated during the first year of life with three doses of a combined vaccine against diphtheria, tetanus, poliomyelitis, and pertussis. A rapid clinical improvement was observed under erythromycin treatment. Other cases of laryngeal diphtheria have been observed. It is important to reverse decreasing vaccinal coverage in Djibouti and to warn incoming travelers of the need to be adequate immunized against diphtheria. Enhanced epidemiologic surveillance of this disease is also needed.

  18. [Identification of heterologous antitoxin in sera of patients with diphtheria].

    PubMed

    Gal'vidis, I A; Burkin, M A; Sviridov, V V

    2008-01-01

    Using immobilized diphtheria toxin and peroxidase conjugate of monoclonal antibodies to light chains of equine immunoglobulin a method of quantification of equine antibodies against diphtheria in sera of patients after serotherapy was developed. The sensitivity of indirect enzyme-linked immunosorbent assay was 0.0005 IU/ml, and coefficient of variation did not exceed 10%. It was shown that in patients with toxic diphtheria heterologous antitoxin is eliminated within 4-6 weeks. Level of anti-diphtheria immunoglobulin under the similar severity of disease and dosage of antitoxin can vary in wide ranges and depends from individual's characteristics.

  19. Physicochemical and immunochemical assays for monitoring consistent production of tetanus toxoid.

    PubMed

    Metz, Bernard; Tilstra, Wichard; van der Put, Robert; Spruit, Nanda; van den Ijssel, Jan; Robert, Jolanda; Hendriksen, Coenraad; Kersten, Gideon

    2013-07-01

    The detoxification of tetanus toxin by formaldehyde is a crucial step in the production of tetanus toxoid. The inactivation results in chemically modified proteins and it determines largely the ultimate efficacy and safety of the vaccine. Currently, the quality of tetanus toxoid lots is evaluated in potency and safety tests performed in animals. As a possible alternative, this article describes a panel of in vitro methods, which provides detailed information about the quality of tetanus toxoid. Ten experimental lots of tetanus toxoid were prepared using increasing concentrations of formaldehyde and glycine to obtain tetanus toxoids having differences in antigenicity, immunogenicity, residual toxicity and protein structure. The structural properties of each individual toxoid were determined using immunochemical and physicochemical methods, including biosensor analysis, ELISA, circular dichroism, TNBS assay, differential scanning calorimetry, fluorescence and SDS-PAGE. The quality of a tetanus toxoid lot can be assessed by these set of analytical techniques. Based on antigenicity, immunogenicity and residual toxicity data, criteria are formulated that tetanus toxoids lot have to meet in order to have a high quality. The in vitro methods are a valuable selection of techniques for monitoring consistency of production of tetanus toxoid, especially for the detoxification process of tetanus toxin.

  20. Development and clinical testing of multivalent vaccines based on a diphtheria-tetanus-acellular pertussis vaccine: difficulties encountered and lessons learned.

    PubMed

    Capiau, Carine; Poolman, Jan; Hoet, Bernard; Bogaerts, Hugues; Andre, Francis

    2003-06-02

    The widespread use of whole-cell pertussis vaccines in the second half of the 20th century have reduced the incidence of the disease significantly. However, in some countries, concerns about the reactogenicity and potential neurological damage associated with whole-cell vaccines led to a decrease in vaccine acceptance and an increase in morbidity and mortality of pertussis in several countries. This prompted the development of less reactogenic acellular pertussis vaccines combined with diphtheria and tetanus toxoids, initially in Japan and later in other countries. In Europe, the improved diphtheria, tetanus and acellular pertussis (DTPa) vaccine was first introduced in March 1994. The pertussis component of this DTPa vaccine, developed by Glaxo SmithKline, consists of pertussis toxoid, filamentous haemagglutinin and pertactin. The vaccine is well tolerated, with a lower incidence of adverse reactions than after administration of whole-cell vaccines. The long-lasting efficacy and effectiveness of DTPa vaccines have been extensively documented and these are now the cornerstone of a large range of combined vaccines including DTPa-hepatitis B (HBV), DTPa-inactivated polio (IPV) and DTPa-HBV-IPV. A lyophilised Haemophilus influenzae type b (Hib) vaccine can be reconstituted with all of these liquid combinations. The introduction of well-tolerated and efficacious DTPa vaccines and their more polyvalent combinations has improved the acceptance and simplified the implementation of childhood immunisation. This paper is a review of the technical and scientific difficulties encountered and the lessons learned over the 10-year period that it took to develop and introduce six multivalent vaccines using the Glaxo SmithKline DTPa as a building block.

  1. Your Child's Immunizations: Diphtheria, Tetanus & Pertussis Vaccine (DTaP)

    MedlinePlus

    ... Year-Old Your Child's Immunizations: Diphtheria, Tetanus & Pertussis Vaccine (DTaP) KidsHealth > For Parents > Your Child's Immunizations: Diphtheria, ... severe burn to prevent tetanus infection. Why the Vaccine Is Recommended Use of the DTaP vaccine has ...

  2. Comparison of five commercial anti-tetanus toxoid immunoglobulin G enzyme-linked immunosorbent assays.

    PubMed

    Perry, A L; Hayes, A J; Cox, H A; Alcock, F; Parker, A R

    2009-12-01

    Five commercially available enzyme-linked immunosorbent assays for the measurement of anti-tetanus toxoid immunoglobulin G (IgG) antibodies were evaluated for performance. The data suggest that there are manufacturer-dependent differences in sensitivity and accuracy for the determination of tetanus toxoid IgG antibodies that could result in different diagnostic interpretations.

  3. Prevalence of diphtheria and tetanus antibodies and circulation of Corynebacterium diphtheriae in São Paulo, Brazil.

    PubMed

    Divino-Goes, K G; Moraes-Pinto, M I de; Dinelli, M I S; Casagrande, S T; Bonetti, T C S; Andrade, P R; Weckx, L Y

    2007-12-01

    The introduction of routine vaccination against tetanus and diphtheria in Brazil has decreased the incidence and changed the epidemiology of both diseases. We then investigated the prevalence of Corynebacterium diphtheriae carrier status and diphtheria and tetanus immunity in São Paulo, Brazil. From November 2001 to March 2003, 374 individuals were tested for the presence of C. diphtheriae in the naso-oropharynx and of serum diphtheria and tetanus antibodies. Participants were all healthy individuals without acute or chronic pathologies and they were stratified by age as follows: 0-12 months and 1-4, 5-9, 10-14, 15-24, 25-39, 40-59, and > or =60 years. Antibodies were assessed using a double-antigen ELISA. C. diphtheriae species were identified by biochemical analysis and toxigenicity was assessed by the Elek test. For diphtheria, full protection (antibodies > or =0.1 IU/mL) was present in 84% of the individuals, 15% had basic protection (antibodies > or =0.01 and <0.1 IU/mL) and 1% were susceptible (antibodies <0.01 IU/mL). Full tetanus protection (antibodies > or =0.1 IU/mL) was present in 79% of the participants, 18% had basic protection (antibodies > or =0.01 and <0.1 IU/mL) and 3% were susceptible (antibodies <0.01 IU/mL). The geometric mean of diphtheria and tetanus antibodies reached the highest values at 5-9 years and decreased until the 40-59-year age range, increasing again in individuals over 60 years. Three participants (0.8%) were carriers of C. diphtheriae, all non-toxigenic strains. The present results demonstrate the clear need of periodic booster for tetanus and diphtheria vaccine in adolescents and adults after primary immunization in childhood.

  4. Adsorption of Toll-Like Receptor 4 Agonist to Alum-Based Tetanus Toxoid Vaccine Dampens Pro-T Helper 2 Activities and Enhances Antibody Responses

    PubMed Central

    Bortolatto, Juliana; Mirotti, Luciana; Rodriguez, Dunia; Gomes, Eliane; Russo, Momtchilo

    2015-01-01

    Aluminum salts gels (alum) are TLR-independent adjuvants and have been used to boost antibody responses in alum-based vaccines such as diphtheria, pertussis, and tetanus toxoid (DPT) triple vaccine. However, the pro-Th2 activity of alum-based vaccine formulations has not been fully appreciated. Here we found that alum-based tetanus toxoid (TT) vaccine was biased toward a Th-2 profile as shown by TT-induced airway eosinophilic inflammation, type 2 cytokine production, and high levels of IgE anaphylactic antibodies. The adsorption into alum of prototypic TLR4 agonists such as lipopolysaccharides (LPS) derived from Escherichia coli consistently dampened TT-induced Th2 activities without inducing IFNγ or Th1-like responses in the lung. Conversely, adsorption of monophosphoryl lipid A (MPLA) extracted from Salmonella minnesota, which is a TIR-domain-containing adapter-inducing interferon-β- (TRIF-) biased TLR4 agonist, was less effective in decreasing Th-2 responses. Importantly, in a situation with antigenic competition (OVA plus TT), TT-specific IgG1 or IgG2a was decreased compared with TT sensitization. Notably, LPS increased the production of IgG1 and IgG2a TT-specific antibodies. In conclusion, the addition of LPS induces a more robust IgG1 and IgG2a TT-specific antibody production and concomitantly decreases Th2-cellular and humoral responses, indicating a potential use of alum/TLR-based vaccines. PMID:26380316

  5. Tellurite resistance: a putative pitfall in Corynebacterium diphtheriae diagnosis?

    PubMed

    dos Santos, Louisy Sanches; Antunes, Camila Azevedo; de Oliveira, Daniel Martins; Sant'Anna, Lincoln de Oliveira; Pereira, José Augusto Adler; Hirata Júnior, Raphael; Burkovski, Andreas; Mattos-Guaraldi, Ana Luíza

    2015-11-01

    Corynebacterium diphtheriae strains continue to circulate worldwide causing diphtheria and invasive diseases, such as endocarditis, osteomyelitis, pneumonia and catheter-related infections. Presumptive C. diphtheriae infections diagnosis in a clinical microbiology laboratory requires a primary isolation consisting of a bacterial culture on blood agar and agar containing tellurite (TeO3(2-)). In this study, nine genome sequenced and four unsequenced strains of C. diphtheriae from different sources, including three samples from a recent outbreak in Brazil, were characterized with respect to their growth properties on tellurite-containing agar. Levels of tellurite-resistance (Te(R)) were evaluated by determining the minimum inhibitory concentrations of potassium tellurite (K2TeO3) and by a viability reduction test in solid culture medium with K2TeO3. Significant differences in Te(R) levels of C. diphtheriae strains were observed independent of origin, biovar or presence of the tox gene. Data indicated that the standard initial screening with TeO3(2-)-selective medium for diphtheria bacilli identification may lead to false-negative results in C. diphtheriae diagnosis laboratories.

  6. Meningococcal groups C and Y and haemophilus B tetanus toxoid conjugate vaccine (HibMenCY-TT; MenHibrix(®)): a review.

    PubMed

    Perry, Caroline M

    2013-05-01

    The meningococcal groups C and Y and Haemophilus b (Hib) tetanus toxoid conjugate vaccine (HibMenCY-TT) contains Neisseria meningitidis serogroup C and Y capsular polysaccharide antigens, and Hib capsular polysaccharide [polyribosyl-ribitol-phosphate (PRP)]. The HibMenCY-TT vaccine is available in the USA for use as active immunization to prevent invasive disease caused by N. meningitidis serogroups C (MenC) and Y (MenY), and Hib in children 6 weeks-18 months of age. HibMenCY-TT is the first meningococcal vaccine available for use in the USA that can be administered to infants as young as 6 weeks of age. In a randomized, controlled, phase III clinical trial, the HibMenCY-TT vaccine, administered to infants at 2, 4, 6 and 12-15 months of age, was immunogenic against MenC and MenY, and met the prespecified criteria for immunogenicity. Anti-PRP antibodies, which have been shown to correlate with protection against Hib invasive disease, were also induced in the infants who received the HibMenCY-TT vaccine, with induced levels of this antibody noninferior to those occurring in the control group of infants who received a Hib tetanus toxoid conjugate vaccine at 2, 4, and 6 months and a single dose of Hib conjugated to N. meningitidis outer membrane protein at 12-15 months. In several randomized, controlled clinical trials, HibMenCY-TT was coadministered with vaccines that are routinely administered to infants and toddlers in the USA. These vaccines included: diphtheria and tetanus toxoids and acellular pertussis adsorbed, hepatitis B (recombinant) and inactivated poliovirus vaccine combined; 7-valent Streptococcus pneumoniae polysaccharide conjugate vaccine; measles, mumps and rubella vaccine; and varicella vaccine. Coadministration of these vaccines did not interfere with the immunogenicity of the HibMenCY-TT vaccine. Similarly, immune responses to the coadministered vaccines were not affected by the HibMenCY-TT vaccine. The tolerability profile of the Hib

  7. Oral vaccination against diphtheria using polyacryl starch microparticles as adjuvant.

    PubMed

    Rydell, Niclas; Sjöholm, Ingvar

    2004-03-12

    Oral vaccination offers the advantage of eliciting both a mucosal and a systemic immune response. This study investigated the use of polyacryl starch microparticles as adjuvant for oral vaccination against diphtheria. Diphtheria toxin or cross-reacting material (CRM197) were covalently conjugated to the microparticles and fed to mice by oral gavage. Investigation of formaldehyde treatment as a means of either detoxifying (diphtheria toxin) or stabilising (CRM197) these formulations were also made. We show that all our formulations given orally or parenterally to mice induced a strong systemic immune response. Only formulations given orally induced a mucosal IgA-response. Furthermore, our formulations given parenterally or orally induced a strong diphtheria toxin-neutralising antibody response.

  8. Efficacy of diphtheria and tetanus vaccination in Gaza, Palestine.

    PubMed

    Al Aswad, I H; Shubair, M E

    2009-01-01

    This study evaluated the effectiveness and usefulness of vaccination against diphtheria and tetanus in different age groups in Gaza, Palestine. Blood samples were collected from 180 children aged <12 years, 90 males and 90 females. Using ELISA methods, the efficacy of vaccination was estimated at 87.8% for diphtheria and 98.3% for tetanus. Mean serum titres varied significantly by age group: for diphtheria 0.24 IU/mL at age 2-4 years, 0.63 IU/mL at 7-8 years and 0.46 IU/mL at 11-12 years, and for tetanus 1.01 IU/mL, 2.63 IU/mL and 1.20 IU/mL respectively. The relatively low antibody titres, especially for diphtheria, suggest the need for a booster dose.

  9. Td Vaccine (Tetanus and Diphtheria): What You Need to Know

    MedlinePlus

    ... best medical care. DIPHTHERIA can cause a thick coating to form in the back of the throat. • ... state health department. • Contact the Centers for Disease Control and Prevention (CDC): - Call 1-800-232-4636 ( ...

  10. Your Child's Immunizations: Diphtheria, Tetanus & Pertussis Vaccine (DTaP)

    MedlinePlus

    ... by toxin-producing bacteria contaminating a wound Pertussis (whooping cough) : a respiratory illness with cold-like symptoms that ... For Parents MORE ON THIS TOPIC Diphtheria Tetanus Whooping Cough (Pertussis) How Can I Comfort My Baby During ...

  11. Recent Outbreaks of Diphtheria in Dibrugarh District, Assam, India

    PubMed Central

    Patgiri, Saurav Jyoti; Saikia, Lahari; Paul, Debosmita

    2016-01-01

    Diphtheria is still a significant child health problem in countries with low immunization coverage. Reports of diphtheria in adult population are also increasing. Here we describe three recent outbreaks of diphtheria in Dibrugarh district, Assam in two consecutive years. The study was undertaken in Assam Medical College & Hospital, Dibrugarh after the diagnosis of two Diphtheria cases in the month of September and October 2015 and another in January 2016. Outbreak investigation was done after defining operational definition and throat swabs were collected from thirty three (33) individuals including three (3) index cases and thirty (30) close contacts. Diagnosis was done by clinical findings, direct microscopy, bacteriological culture and in-house designed multiplex Polymerase Chain Reaction (PCR) of the isolates for the expression of Corynebacterium diphtheriae specific rpoB gene and tox gene. Out of the 10 confirmed cases, 2 and 7 were in the first two outbreaks while only one in the third outbreak respectively. All the cases were of age > 10 years, unimmunized or partially immunized. The overall mortality was 20%. PCR results revealed all the culture positive isolates to be tox gene positive. Diphtheria is a resurgent problem in our region with a significant age shift towards adult. PMID:27630847

  12. Immune responses of Asian elephants (Elephas maximus) to commercial tetanus toxoid vaccine.

    PubMed

    Lindsay, William A; Wiedner, Ellen; Isaza, Ramiro; Townsend, Hugh G G; Boleslawski, Maria; Lunn, D P

    2010-02-15

    Although captive elephants are commonly vaccinated annually against tetanus using commercially available tetanus toxoid vaccines marketed for use in horses and livestock, no data exists to prove that tetanus toxoid vaccination produces measurable antibody titers in elephants. An ELISA test was created to measure antibody responses to tetanus toxoid vaccinations in 22 Asian elephants ranging in age from 24 to 56 years (mean age 39 years) over a 7-month period. All animals had been previously vaccinated with tetanus toxoid vaccine, with the last booster administered 4 years before the start of the study. The great majority of elephants had titers prior to booster vaccination, and following revaccination all elephants demonstrated anamnestic increases in titers, indicating that this species does respond to tetanus vaccination. Surprisingly older animals mounted a significantly higher response to revaccination than did younger animals.

  13. Corynebacterium diphtheriae: genome diversity, population structure and genotyping perspectives.

    PubMed

    Mokrousov, Igor

    2009-01-01

    The epidemic re-emergence of diphtheria in Russia and the Newly Independent States (NIS) of the former Soviet Union in the 1990s demonstrated the continued threat of this thought to be rare disease. The bacteriophage encoded toxin is a main virulence factor of Corynebacterium diphtheriae, however, an analysis of the first complete genome sequence of C. diphtheriae revealed a recent acquisition of other pathogenicity factors including iron-uptake systems, adhesins and fimbrial proteins as indeed this extracellular pathogen has more possibilities for lateral gene transfer than, e.g., its close relative, mainly intracellular Mycobacterium tuberculosis. C. diphtheriae appears to have a phylogeographical structure mainly represented by area-specific variants whose circulation is under strong influence of human host factors, including health control measures, first of all, vaccination, and social economic conditions. This framework core population structure may be challenged by importation of the endemic and eventually toxigenic strains from new areas thus leading to localized or large epidemics caused directly by imported strains or by bacteriophage-lysogenized indigenous strains converted into toxin production. A feature of C. diphtheriae co-existence with humans is its periodicity: following large epidemic in the 1990s, the present period is marked by increasing heterogeneity of the circulating populations whereas re-emergence of new toxigenic variants along with persistent circulation of invasive non-toxigenic strains appear alarming. To identify and rapidly monitor subtle changes in the genome structure at an infraclonal level during and between epidemics, portable and discriminatory typing methods of C. diphtheriae are still needed. In this view, CRISPRs and minisatellites are promising genomic markers for development of high-resolution typing schemes and databasing of C. diphtheriae.

  14. Purification and structural characterization of siderophore (corynebactin) from Corynebacterium diphtheriae.

    PubMed

    Zajdowicz, Sheryl; Haller, Jon C; Krafft, Amy E; Hunsucker, Steve W; Mant, Colin T; Duncan, Mark W; Hodges, Robert S; Jones, David N M; Holmes, Randall K

    2012-01-01

    During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin.

  15. Purification and Structural Characterization of Siderophore (Corynebactin) from Corynebacterium diphtheriae

    PubMed Central

    Zajdowicz, Sheryl; Haller, Jon C.; Krafft, Amy E.; Hunsucker, Steve W.; Mant, Colin T.; Duncan, Mark W.; Hodges, Robert S.; Jones, David N. M.; Holmes, Randall K.

    2012-01-01

    During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin. PMID:22514641

  16. Respiratory diphtheria in an asylum seeker from Afghanistan arriving to Finland via Sweden, December 2015.

    PubMed

    Sane, Jussi; Sorvari, Tiina; Widerström, Micael; Kauma, Heikki; Kaukoniemi, Ulla; Tarkka, Eveliina; Puumalainen, Taneli; Kuusi, Markku; Salminen, Mika; Lyytikäinen, Outi

    2016-01-01

    In December 2015, an asylum seeker originating from Afghanistan was diagnosed with respiratory diphtheria in Finland. He arrived in Finland from Sweden where he had already been clinically suspected and tested for diphtheria. Corynebacterium diphtheriae was confirmed in Sweden and shown to be genotypically and phenotypically toxigenic. The event highlights the importance of early case detection, rapid communication within the country and internationally as well as preparedness plans of diphtheria antitoxin availability.

  17. Sequence Analysis of Toxin Gene-Bearing Corynebacterium diphtheriae Strains, Australia.

    PubMed

    Doyle, Christine J; Mazins, Adam; Graham, Rikki M A; Fang, Ning-Xia; Smith, Helen V; Jennison, Amy V

    2017-01-01

    By conducting a molecular characterization of Corynebacterium diphtheriae strains in Australia, we identified novel sequences, nonfunctional toxin genes, and 5 recent cases of toxigenic cutaneous diphtheria. These findings highlight the importance of extrapharyngeal infections for toxin gene-bearing (functional or not) and non-toxin gene-bearing C. diphtheriae strains. Continued surveillance is recommended.

  18. Bloodstream infection caused by nontoxigenic Corynebacterium diphtheriae in an immunocompromised host in the United States.

    PubMed

    Wojewoda, Christina M; Koval, Christine E; Wilson, Deborah A; Chakos, Mary H; Harrington, Susan M

    2012-06-01

    Corynebacterium species are well-known causes of catheter-related bloodstream infections. Toxigenic strains of Corynebacterium diphtheriae cause respiratory diphtheria. We report a bloodstream infection caused by a nontoxigenic strain of C. diphtheriae and discuss the epidemiology, possible sources of the infection, and the implications of rapid species identification of corynebacteria.

  19. Sequence Analysis of Toxin Gene–Bearing Corynebacterium diphtheriae Strains, Australia

    PubMed Central

    Mazins, Adam; Graham, Rikki M.A.; Fang, Ning-Xia; Smith, Helen V.; Jennison, Amy V.

    2017-01-01

    By conducting a molecular characterization of Corynebacterium diphtheriae strains in Australia, we identified novel sequences, nonfunctional toxin genes, and 5 recent cases of toxigenic cutaneous diphtheria. These findings highlight the importance of extrapharyngeal infections for toxin gene–bearing (functional or not) and non–toxin gene–bearing C. diphtheriae strains. Continued surveillance is recommended. PMID:27983494

  20. Fibrinogen binds to nontoxigenic and toxigenic Corynebacterium diphtheriae strains.

    PubMed

    Sabbadini, Priscila Soares; Genovez, Marcia Rocha Novais; Silva, Cecília Ferreira da; Adelino, Thelma Lúcia Novaes; Santos, Cintia Silva dos; Pereira, Gabriela Andrade; Nagao, Prescilla Emy; Dias, Alexandre Alves de Souza de Oliveira; Mattos-Guaraldi, Ana Luiza; Hirata Júnior, Raphael

    2010-08-01

    The production of fibrinous exudates may play an important role in determining the outcome of bacterial infection. Although pseudomembrane formation is a characteristic feature of diphtheria, little is known about the fibrinogen (Fbn)-binding properties of Corynebacterium diphtheriae strains and the influence of the gene that codes for diphtheria toxin (tox gene) in this process. In this study we demonstrated the ability of C. diphtheriae strains to bind to Fbn and to convert Fbn to fibrin. Bacterial interaction with rabbit plasma was evaluated by both slide and tube tests. Interaction of microorganisms with human Fbn was evaluated by both enzyme linked immunosorbent assay (ELISA) and fluorescein isothiocyanate-conjugated (FITC) Fbn binding assays. Nontoxigenic and toxigenic strains formed bacterial aggregates in the presence of plasma in the slide tests. The ability to convert Fbn to a loose web of fibrin in the plasma solution in the tube tests appeared to be a common characteristic of the species, including strains that do not carry the tox gene. Fbn binding to C. diphtheriae strains occurred at varying intensities, as demonstrated by the FITC-Fbn and ELISA binding assays. Our data suggest that the capacity to bind to Fbn and to convert Fbn to fibrin may play a role in pseudomembrane formation and act as virulence determinants of both nontoxigenic and toxigenic strains.

  1. [Production and characteristics of monoclonal antibodies to the diphtheria toxin].

    PubMed

    Valiakina, T I; Lakhtina, O E; Komaleva, R L; Simonova, M A; Samokhvalova, L V; Shoshina, N S; Kalinina, N A; Rubina, A Iu; Filippova, M A; Vertiev, Iu V; Grishin, E V

    2009-01-01

    Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

  2. Immunogenicity and safety of diphtheria-tetanus vaccine in adults.

    PubMed

    Choi, Jung-Hyun; Choo, Eun Ju; Huh, Aejung; Choi, Su-Mi; Eom, Joong Sik; Lee, Jin Seo; Park, Sun Hee; Kang, Jin Han

    2010-12-01

    This study was conducted to evaluate the immunogenicity and safety of diphtheria-tetanus (Td) vaccine in adults over 40 yr old who had never received a diphtheria-tetanus-pertussis (DTP) vaccination. A total of 242 subject completed three-doses of Td vaccination and subsequent assays for immunogenicity. Before vaccination, 33.9% and 96.7% participants showed antibody levels of diphtheria and tetanus, respectively, which were below protective level (<0.1 U/mL). After the first dose of Td vaccine, 92.6% and 77.6% of subjects gained protective antibody concentrations (≥ 0.1 U/mL) for diphtheria and tetanus, with an increase to 99.6% and 100% after the third dose. Local and systemic adverse events occurred in 37.9% and 15.5% of the subjects. No serious adverse event requiring an unscheduled hospital visit occurred. In conclusion, three-doses of Td vaccination to unimmunized adults are safe and effective in inducing protective immunity against diphtheria and tetanus.

  3. Immunogenicity and safety of a combined diphtheria, tetanus, acellular pertussis, and inactivated poliovirus vaccine (DTaP-IPV) compared to separate administration of standalone DTaP and IPV vaccines: a randomized, controlled study in infants in the Republic of Korea.

    PubMed

    Lee, Soo Young; Hwang, Hui Sung; Kim, Jong Hyun; Kim, Hyun Hee; Lee, Hyun Seung; Chung, Eun Hee; Park, Su Eun; Ma, Sang Hyuk; Chang, Jin Keun; Guitton, Fabrice; Ortiz, Esteban; Kang, Jin Han

    2011-02-11

    This randomized trial enrolled 442 infants in the Republic of Korea to assess the immunogenicity and safety of a combined diphtheria, tetanus, acellular pertussis, and inactivated poliovirus vaccine (DTaP-IPV; Tetraxim™) for primary vaccination at 2, 4 and 6 months of age compared with DTaP and IPV vaccines given separately. Immunogenicity was high in both groups; seroprotection and seroconversion rates of the combined vaccine (Group A) were non-inferior to the control vaccines (Group B). All subjects were seroprotected against poliovirus types 1, 2 and 3 (≥ 81/dil) and anti-diphtheria (≥ 0.01 IU/mL); 99.0% were seroprotected against tetanus (≥ 0.1 IU/mL). At least 93.6% had anti-diphtheria antibody titers ≥ 0.1 IU/mL. Anti-pertussis toxoid (PT) and anti-filamentous haemagglutinin (FHA) seroconversion (≥ 4-fold increase in antibody titer) rates were 96.6% and 94.4% for Group A, 92.2% and 78.4% for Group B. Most solicited reactions occurred within 4 days of vaccination, resolved within 3 days and were mild. Severe solicited reactions occurred after ≤ 0.5% of doses in Group A and ≤ 0.9% in Group B. No withdrawals occurred because of adverse events. The DTaP-IPV combined vaccine given at 2, 4, and 6 months of age was well tolerated; immunogenicity was similar to the control vaccines.

  4. The early primary immune response to adsorbed tetanus toxoid in man

    PubMed Central

    Maclennan, R.; Levine, L.; Newell, K. W.; Edsall, G.

    1973-01-01

    A quantitative study was performed to determine the effect of toxoid concentration and aluminium salt concentration on the primary immune response (PIR) and the secondary response induced by tetanus toxoid in human volunteers. Four toxoid preparations having 5-fold differences in toxoid concentration, aluminium salt concentration, or both, were administered to four comparable groups of human volunteers. Antitoxin titres in the serum of each volunteer were determined at intervals. The PIR was found to be a function of the antigen concentration, the mineral concentration, and the interaction of both. The secondary response was a function of the antigen concentration; increase in mineral adjuvant concentration had no significant effect. The data suggested that the higher the post-secondary response, the slower the rate of decline over the ensuing 10 months. The distribution of primary responses at day 28 tended to be bimodal. The response to the best preparation suggested that a single-dose toxoid might be developed to immunize populations that may be difficult to retrieve for multiple injections. PMID:4548388

  5. Randomized trial on the safety, tolerability, and immunogenicity of MenACWY-CRM, an investigational quadrivalent meningococcal glycoconjugate vaccine, administered concomitantly with a combined tetanus, reduced diphtheria, and acellular pertussis vaccine in adolescents and young adults.

    PubMed

    Gasparini, Roberto; Conversano, Michele; Bona, Gianni; Gabutti, Giovanni; Anemona, Alessandra; Dull, Peter M; Ceddia, Francesca

    2010-04-01

    This study evaluated the safety, tolerability, and immunogenicity of an investigational quadrivalent meningococcal conjugate vaccine, MenACWY-CRM, when administered concomitantly with a combined tetanus, reduced diphtheria, and acellular pertussis (Tdap) vaccine, in subjects aged 11 to 25 years. Subjects received either MenACWY-CRM and Tdap, MenACWY-CRM and saline placebo, or Tdap and saline placebo. No significant increase in reactogenicity and no clinically significant vaccine-related adverse events (AEs) occurred when MenACWY-CRM and Tdap were administered concomitantly. Similar immunogenic responses to diphtheria, tetanus, and meningococcal (serogroups A, C, W-135, and Y) antigens were observed, regardless of concomitant vaccine administration. Antipertussis antibody responses were comparable between vaccine groups for filamentous hemagglutinin and were slightly lower, although not clinically significantly, for pertussis toxoid and pertactin when the two vaccines were administered concomitantly. These results indicate that the investigational MenACWY-CRM vaccine is well tolerated and immunogenic and that it can be coadministered with Tdap to adolescents and young adults.

  6. Co-administration of a novel Haemophilus influenzae type b and Neisseria meningitidis serogroups C and Y-tetanus toxoid conjugate vaccine does not interfere with the immune response to antigens contained in infant vaccines routinely used in the United States.

    PubMed

    Marshall, Gary S; Marchant, Colin D; Blatter, Mark; Friedland, Leonard R; Aris, Emmanuel; Miller, Jacqueline M

    2011-02-01

    An investigational combined Haemophilus influenzae type b (Hib) and Neisseria meningitidis serogroups C and Y tetanus toxoid conjugate vaccine (HibMenCY-TT) has been developed to protect infants from invasive disease caused by Hib and these meningococcal serogroups without adding injections to the immunization schedule. Incorporation of this novel vaccine into the US vaccination schedule will require demonstration of a lack of immunologic interference with other routine pediatric vaccines. This study assessed the immune response to 7-valent pneumococcal conjugate vaccine (PCV7) and combined diphtheria-tetanus-acellular pertussis-hepatitis B-inactivated poliovirus vaccine (DTaP-HepB-IPV) when separately co-administered with HibMenCY-TT as compared to a US-licensed H. influenzae type b tetanus toxoid conjugate vaccine (Hib-TT) at 2, 4, 6 (N=606) and 12-15 months of age (N=366). HibMenCY-TT was non-inferior to Hib-TT in terms of antibody responses to all Streptococcus pneumoniae serotypes contained in PCV7 and the diphtheria, tetanus, pertussis, hepatitis B and poliovirus antigens contained in DTaP-HepB-IPV one month after the third vaccine dose, and the anti-tetanus geometric mean antibody concentration (GMC) was significantly higher in the HibMenCY-TT group than in the Hib-TT group. In an exploratory analysis, no significant differences in the proportion of subjects with anti-pneumococcal antibody concentrations ≥0.2 µg/ml or anti-pneumococcal GMC were seen between the two groups after the fourth vaccine dose. A schedule of HibMenCY-TT given concomitantly with PCV7 and DTaP-HepB-IPV would be expected to protect infants against all of the targeted diseases.

  7. Protection against avian necrotic enteritis after immunisation with NetB genetic or formaldehyde toxoids.

    PubMed

    Fernandes da Costa, Sérgio P; Mot, Dorien; Bokori-Brown, Monika; Savva, Christos G; Basak, Ajit K; Van Immerseel, Filip; Titball, Richard W

    2013-08-20

    NetB (necrotic enteritis toxin B) is a recently identified β-pore-forming toxin produced by Clostridium perfringens. This toxin has been shown to play a major role in avian necrotic enteritis. In recent years, a dramatic increase in necrotic enteritis has been observed, especially in countries where the use of antimicrobial growth promoters in animal feedstuffs has been banned. The aim of this work was to determine whether immunisation with a NetB toxoid would provide protection against necrotic enteritis. The immunisation of poultry with a formaldehyde NetB toxoid or with a NetB genetic toxoid (W262A) resulted in the induction of antibody responses against NetB and provided partial protection against disease.

  8. Stabilization of tetanus toxoid formulation containing aluminium hydroxide adjuvant against freeze-thawing.

    PubMed

    Solanki, Vipul A; Jain, Nishant K; Roy, Ipsita

    2011-07-29

    Exposure to subzero temperature leads to loss of vaccine potency. This can happen due to degradation of adjuvant surface and/or inactivation of the antigen. When adsorbed on aluminium hydroxide and subjected to freeze-thawing, tetanus toxoid was desorbed from the gel matrix and the preparation was found to lose its antigenicity. Analyses showed that the gel particles were denatured after freezing. When freeze-thawing was carried out in the presence of glucose, sorbitol and arginine, the degradation of gel particles was inhibited. A higher fraction of the protein could be retained on the gel. However, the antigenicity of these preparations was quite low. In the presence of trehalose, the protein could be partially retained on aluminium hydroxide. Being a cryoprotectant, trehalose was also able to inhibit the freezing-induced denaturation of tetanus toxoid, which resulted in retention of antigenicity of the adjuvanted toxoid.

  9. Stabilization of tetanus toxoid formulation containing aluminium hydroxide adjuvant against agitation.

    PubMed

    Solanki, Vipul A; Jain, Nishant K; Roy, Ipsita

    2012-02-28

    The aggregation of tetanus toxoid leads to reduced bioavailability of the vaccine and failure of immunization programmes in many parts of the globe. One of the main reasons for denaturation and aggregation of tetanus toxoid formulations is agitation of the protein during transport. We have identified that agitation leads to collapse of the gel matrix of aluminium hydroxide which is used as an adjuvant in these preparations. This results in desorption of the toxoid from the matrix, which then loses its antigenicity due to agitation-induced denaturation of the protein. We show that incorporation of some compatible osmolytes like sorbitol, glucose and arginine, but not trehalose, is able to protect the adjuvant matrix from degradation, and retain the integrity of the vaccine preparation in terms of its antigenicity.

  10. Specific detection of tetanus toxoid using an aptamer-based matrix.

    PubMed

    Modh, Harshvardhan B; Bhadra, Ankan K; Patel, Kinjal A; Chaudhary, Rajeev K; Jain, Nishant K; Roy, Ipsita

    2016-11-20

    Batch-to-batch variation of therapeutic proteins produced by biological means requires rigorous monitoring at all stages of the production process. A large number of animals are employed for risk assessment of biologicals, which has low ethical and economic acceptability. Research is now focussed on the validation of in vitro and ex vivo tests to replace live challenges. Among in vitro methods, enzyme-linked immunosorbent assay (ELISA) is considered to be the gold standard for estimation of integrity of tetanus toxoid. ELISA utilizes antibodies for detection, which, because of their biological origin and limited modifiability, may have low stability and result in irreproducibility. We have developed a method using highly specific and selective RNA aptamers for detection of tetanus toxoid. Using displacement assay, we first identified aptamers which bind to different aptatopes on the surface of the toxoid. Pairs of these aptamers were employed as capture-detection ligands in a sandwich-ALISA (aptamer-linked immobilized sorbent assay) format. The binding efficiency was confirmed by the fluorescence intensity in each microtire plate well. Using aptamers alone, detection of tetanus toxoid was possible with the same level of sensitivity as antibody. Aptamers were also used in the capture ALISA format. Adjuvanted tetanus toxoid was subjected to accelerated stress testing, including thermal, mechanical and freeze-thawing stress conditions. The loss in antigenicity of the preparation determined by ALISA in each case was found to be similar to that determined by conventional ELISA. Thus, it is possible to replace antibodies with aptamers to develop a more robust detection tool for tetanus toxoid.

  11. The draft genome sequence of Corynebacterium diphtheriae bv. mitis NCTC 3529 reveals significant diversity between the primary disease-causing biovars.

    PubMed

    Sangal, Vartul; Tucker, Nicholas P; Burkovski, Andreas; Hoskisson, Paul A

    2012-06-01

    We report the draft genome of the human pathogen Corynebacterium diphtheriae bv. mitis NCTC 3529. This is the first C. diphtheriae bv. mitis strain to be sequenced and reveals significant differences from the other primary biovar, C. diphtheriae bv. gravis.

  12. Elevated levels of maternal anti-tetanus toxin antibodies do not suppress the immune response to a Haemophilus influenzae type b polyribosylphosphate-tetanus toxoid conjugate vaccine.

    PubMed Central

    Panpitpat, C.; Thisyakorn, U.; Chotpitayasunondh, T.; Fürer, E.; Que, J. U.; Hasler, T.; Cryz, S. J.

    2000-01-01

    Reported are the effects of elevated levels of anti-tetanus antibodies on the safety and immune response to a Haemophilus influenzae type b polyribosylphosphate (PRP)-tetanus toxoid conjugate (PRP-T) vaccine. A group of Thai infants (n = 177) born to women immunized against tetanus during pregnancy were vaccinated with either a combined diphtheria-tetanus-pertussis (DTP) PRP-T vaccine or DTP and a PRP-conjugate vaccine using Neisseria meningitidis group B outer-membrane proteins as a carrier (PedVax HIB). Although most infants possessed high titres (> 1 IU/ml) of anti-tetanus antibodies, the DTP-PRP-T combined vaccine engendered an excellent antibody response to all vaccine components. In both vaccine groups > 98% of infants attained anti-PRP antibody titres > or = 0.15 microgram/ml. The geometric mean anti-PRP antibody titres were 5.41 micrograms/ml and 2.1 micrograms/ml for infants immunized with three doses of PRP-T versus two doses of PedVax HIB vaccines, respectively (P < 0.005). Similarly, the proportion of infants who achieved titres > or = 1 microgram/ml was higher in the PRP-T group (87.8%) than in the group immunized with PedVax HIB (74.2%) (P = 0.036). A subgroup analysis showed that there was no significant difference in the anti-PRP antibody response for infants exhibiting either < 1 IU of anti-tetanus antibody per millilitre or > or = 1 IU/ml at baseline. These finding indicate that pre-existing anti-carrier antibody does not diminish the immune response to the PRP moiety. All infants possessed protective levels of anti-D and anti-T antibody levels after immunization. PMID:10812736

  13. Late-onset prosthetic valve endocarditis caused by nontoxigenic Corynebacterium diphtheriae.

    PubMed

    El-Hazmi, Malak M

    2015-08-29

    In developed countries, Corynebacterium diphtheriae infection is rare due to efficient immunization programs. However, cases of nontoxigenic strains of C. diphtheriae infections, including endocarditis, have been reported recently. Although the incidence remains low, these infections are associated with high morbidity and mortality. This report describes the first and atypical case of bacteremia and endocarditis caused by nontoxigenic C. diphtheriae var. gravis after introduction of immunization in the Kingdom of Saudi Arabia (KSA).

  14. Comparison of a multiplex flow cytometric assay with enzyme-linked immunosorbent assay for auantitation of antibodies to tetanus, diphtheria, and Haemophilus influenzae Type b.

    PubMed

    Pickering, Jerry W; Martins, Thomas B; Schroder, M Carl; Hill, Harry R

    2002-07-01

    We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes. By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (> or = 0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (> or = 0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (> or = 1.0 microg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex. The correlations (R(2)) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration. Both methods detected strong postvaccination responses. The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time.

  15. Identification of Corynebacterium diphtheriae gene involved in adherence to epithelial cells.

    PubMed

    Kolodkina, Valentina; Denisevich, Tatyana; Titov, Leonid

    2011-03-01

    Corynebacterium diphtheriae the causative pathogen of human diphtheria infects the nasopharynx or skin. Although diphtheria has been extensively studied, little is known about the two key aspects of C. diphtheriae invasiveness: colonization and invasion. The role of adhesive properties in establishing the infection of C. diphtheriae strains, independent of toxin production, still needs to be clarified. In this study, we describe a novel gene involved in adherence to epithelial cells. Transformation of C. diphtheriae 225, biotype gravis, ribotype St-Petersburg by EZ:TN(KAN-2)Tnp Transposome was undertaken. A C. diphtheriae 225 Tn5 insertion library of 2800 mutants was created. Five hundred and eighty five transformants were qualitatively screened for reduced adherence to HEp-2 cells by an adherence assay. One mutant strain consistently exhibiting 15.2% of the wild-type adherence was isolated. The DNA flanking the transposon was identified by inverse PCR and subsequent sequencing. The disrupted gene was 94% identical to the C. diphtheriae DIP1621 gene that belongs to unclassified genes. In conclusion, the disruption of the C. diphtheriae DIP1621 gene led to decreased adherence to epithelial cells; its exact function remains to be established.

  16. Process optimization for an industrial-scale production of Diphtheria toxin by Corynebacterium diphtheriae PW8.

    PubMed

    Suwanpatcharakul, Maethichai; Pakdeecharoen, Chompunut; Visuttitewin, Supitcha; Pesirikan, Norapath; Chauvatcharin, Somchai; Pongtharangkul, Thunyarat

    2016-11-01

    In this study, several parameters affecting the toxin production of Corynebacterium diphtheriae Parke Williams 8 (PW8) were investigated in detail. The comparison studies of amino acid profile in NZ Amine A-based medium (NZ medium) and beef digest-based medium (BD medium) suggested that an insufficient supply of amino acids was not responsible for low toxin yield observed in NZ medium. Supplementation of additional amino acids and growth promoting nutrient (in a form of yeast extract) into NZ medium enhanced only cell growth but not toxin production. Thus, BD medium was selected as the most suitable base medium for toxin production as it gave a significantly higher limit of flocculation (93 ± 0 Lf/ml) than NZ medium (46 ± 0 Lf/ml). Interestingly, a supplementation of 0.2% YE into BD medium resulted in a significant increase in growth as well as toxin production (235 ± 5 Lf/ml). In conclusion, consistently high toxin titer (174-239 Lf/ml) could be obtained from BD medium at a 5 L-scale production as long as 1) the protein content of BD medium was at least 24 g/L, 2) the iron content was below 0.15 ppm and 3) 0.2% YE was supplemented into the medium.

  17. Immunological evaluation of meningococcal group C polysaccharide-tetanus toxoid conjugate in mice.

    PubMed Central

    Beuvery, E C; van Delft, R W; Miedema, F; Kanhai, V; Nagel, J

    1983-01-01

    Neisseria meningitidis group C polysaccharide-tetanus toxoid conjugate was prepared to obtain the polysaccharide component in a thymus-dependent form and to preserve the immunogenic properties of the tetanus toxoid component. Biochemical and immunochemical analyses of this conjugate revealed that (i) it was composed of equal amounts of polysaccharide and protein; (ii) the antigenic activity of the polysaccharide component was greatly reduced; (iii) it contained about 10% free polysaccharide; (iv) the composition was not homogeneous; and (v) only 5% of the tetanus toxoid component was present at the surface of the conjugate molecules. In this study, the influence of these characteristics on the antibody response to both components in mice was investigated. The dose-response relationship, the persistence of antibodies, a possible antigenic competition, and the specificities of the antibodies induced were also studied. Our data suggest that the conjugate behaves as a pronounced thymus-dependent antigen, that the tetanus toxoid component is more immunogenic at lower dosages (0.8 and 20 ng) than equivalent doses of tetanus vaccine, that the presence of free polysaccharide does not influence the induction of antibodies to polysaccharide by the conjugate, and that no antibodies to new structures in the conjugate are induced. These characteristics favor the application of this conjugate as a vaccine for human use. PMID:6409811

  18. Potency against enterotoxemia of a recombinant Clostridium perfringens type D epsilon toxoid in ruminants.

    PubMed

    Lobato, Francisco C F; Lima, Catarina G R D; Assis, Ronnie A; Pires, Prhiscylla S; Silva, Rodrigo O S; Salvarani, Felipe M; Carmo, Anderson O; Contigli, Christiane; Kalapothakis, Evanguedes

    2010-08-31

    Enterotoxemia, a disease that affects domestic ruminants, is caused mainly by the epsilon toxin from Clostridium perfringens type D. Its eradication is virtually impossible, control and prophylaxis are based on systematic vaccination of herds with epsilon toxoids that are efficient in inducing protective antibody production. The use of recombinant toxins is one of the most promising of these strategies. This work evaluates the potency of a Cl. perfringens type D epsilon toxoid expressed by Escherichia coli administered to goats, sheep, and cattle. The etx gene was cloned into the pET-11a plasmid of E. coli strain BL21 to produce the recombinant toxin. Rabbits (n=8), goats, sheep, and cattle (n=5 for each species) were immunized with 0.2mg of the insoluble recombinant protein fraction to evaluate vaccine potency of the epsilon toxoid studied. Antibody titers were 40, 14.3, 26, and 13.1 IU/mL in the rabbit, goat, sheep, and cattle serum pools, respectively. The epsilon toxoid produced and tested in this work is adequate for immunization of ruminants against enterotoxemia.

  19. CD4 T-helper cell cytokine phenotypes and antibody response following tetanus toxoid booster immunization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Routine methods for enumerating antigen-specific T-helper cells may not identify low-frequency phenotypes such as Th2 cells. We compared methods of evaluating such responses to identify tetanus toxoid- (TT) specific Th1, Th2, Th17 and IL10+ cells. Eight healthy subjects were given a TT booster vacci...

  20. Tetanus toxoid IgE may be useful in predicting allergy during childhood.

    PubMed

    Ciprandi, G; De Amici, M; Quaglini, S; Labò, E; Castellazzi, A M; Miraglia Del Giudice, M; Marseglia, A; Bianchi, L; Moratti, R; Marseglia, G L

    2012-01-01

    Hypersensitivity reactions after immunization with tetanus toxoid are occasionally observed in atopic and non-atopic individuals. High IgE levels in infancy may predict subsequent allergy. The aims of this study were: i) to evaluate the role of specific IgE to tetanus toxoid in children in response to tetanus immunization and the possible factors associated with specific IgE levels, and ii) to investigate the correlation between specific IgE levels to tetanus toxoid and the late development of allergy (up to 12 years). Initially, 278 healthy infants (152 males and 126 females, aged 12 months) living in an urban city were screened for serum total IgE and specific IgE to tetanus toxoid, after having obtained informed consent from parents. After 12 years, 151 children could be evaluated. Total IgE summed with tetanus specific IgE were significantly associated with allergy at 12 years. In conclusion, this study demonstrates that serum total IgE and tetanus specific IgE may be predictive of subsequent allergy onset.

  1. Safety and Immunogenicity of Tetanus Diphtheria and Acellular Pertussis (Tdap) Immunization During Pregnancy in Mothers and Infants: A Randomized Clinical Trial

    PubMed Central

    Munoz, Flor M.; Bond, Nanette H.; Maccato, Maurizio; Pinell, Phillip; Hammill, Hunter A.; Swamy, Geeta K.; Walter, Emmanuel B.; Jackson, Lisa A.; Englund, Janet A.; Edwards, Morven S.; Healy, C. Mary; Petrie, Carey R.; Ferreira, Jennifer; Goll, Johannes B.; Baker, Carol J.

    2015-01-01

    Importance Maternal immunization with tetanus toxoid and reduced diphtheria toxoid acellular pertussis (Tdap) vaccine could prevent infant pertussis. The effect of vaccine-induced maternal antibodies on infant responses to diphtheria and tetanus toxoids acellular pertussis (DTaP) immunization is unknown. Objective To evaluate the safety and immunogenicity of Tdap immunization during pregnancy and its effect on infant responses to DTaP. Design, Setting and Participants Phase I, randomized, double-masked, placebo-controlled clinical trial conducted in private (Houston) and academic (Durham, Seattle) obstetric practices from 2008 to 2012. Forty eight healthy 18–45 year-old pregnant women received Tdap (n=33) or placebo (n=15) at 30–32 weeks’ gestation with cross-over Tdap immunization postpartum. Interventions Tdap vaccination at 30–32 weeks’ gestation or post-partum. Outcome Measures Primary: Maternal and infant adverse events, pertussis illness and infant growth and development (Bayley-III screening test) until 13 months of age. Secondary: Antibody concentrations in pregnant women before and 4 weeks after Tdap immunization or placebo, at delivery and 2 months postpartum, and in infants at birth, 2 months, and after the third (7 months) and fourth (13 months) doses of DTaP. Results All participants delivered healthy newborns. No Tdap-associated serious adverse events occurred in women or infants. Injection site reactions after Tdap immunization were reported in 78.8% (95% CI: 61.1%, 91.0%) and 80% (CI: 51.9%, 95.7%) pregnant and postpartum women, respectively. Injection site pain was the predominant symptom. Systemic symptoms were reported in 36.4% (CI: 20.4%, 54.9%) and 73.3% (CI: 44.9%, 92.2%) pregnant and postpartum women, respectively. Malaise and myalgia were most common. Growth and development were similar in both infant groups. No cases of pertussis occurred. Significantly higher concentrations of pertussis antibodies were measured at delivery in

  2. Safety and immunogenicity of a meningococcal (Groups A, C, Y, W-135) polysaccharide diphtheria toxoid conjugate vaccine in healthy children aged 2 to 10 years in Chile.

    PubMed

    Lagos, Rosanna; Papa, Thomas; Muñoz, Alma; Ryall, Robert; Pina, Miriam; Bassily, Ehab

    2005-01-01

    Immune responses to meningococcal conjugate (Menactra; MCV-4) and plain polysaccharide (Menomune-A/C/Y/W-135; PSV-4) vaccines against serogroups A, C, Y, and W-135 were assessed in 220 of 1037 Chilean children aged 2 to 10 years participating in a comparative safety trial. Both vaccines were generally well tolerated. Geometric mean serum bactericidal antibody (SBA) titers 28 days postvaccination were comparable in both groups for all four serogroups. Seroconversion was evident in > 97% of MCV-4 and > 90% of PSV-4 vaccinees who tested seronegative at baseline. Menactra safely induced broad and robust immune responses against serogroups A, C, Y and W-135 in this population.

  3. Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae.

    PubMed

    Wesener, Darryl A; Levengood, Matthew R; Kiessling, Laura L

    2017-02-17

    The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis In each species, the galactan is constructed from uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.

  4. Plasticity of Corynebacterium diphtheriae pathogenicity islands revealed by PCR.

    PubMed

    Soares, S C; Dorella, F A; Pacheco, L G C; Hirata, R; Mattos-Guaraldi, A L; Azevedo, V; Miyoshi, A

    2011-06-28

    Despite the existence of a vaccine against diphtheria, this disease remains endemic and is reemerging in several regions due to many factors, including variations in genes coding for virulence factors. One common feature of virulence factors is their high concentration in pathogenicity islands (PAIs), very unstable regions acquired via horizontal gene transfer, which has lead to the emergence of various bacterial pathogens. The 13 putative PAIs in Corynebacterium diphtheriae NCTC 13129 and the reemergence of this disease point to the great variability in the PAIs of this species, which may reflect on bacterial life style and physiological versatility. We investigated the relationships between the large number of PAIs in C. diphtheriae and the possible implications of their plasticity in virulence. The GenoFrag software was used to design primers to analyze the genome plasticity of two pathogenicity islands of the reference strain (PiCds 3 and 8) in 11 different strains. We found that PiCd 3 was absent in only two strains, showing genes playing putative important roles in virulence and that only one strain harbored PiCd 8, due to its location in a putative "hotspot" for horizontal gene transfer events.

  5. 75 FR 7281 - Pediatric Advisory Committee; Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-18

    ... Inactivated Poliovirus Vaccine), Pentacel [Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed, Inactivated Poliovirus and Haemophilus b Conjugate (Tetanus Toxoid Conjugate) Vaccine], and...

  6. Induction of the NFκ-B signal transduction pathway in response to Corynebacterium diphtheriae infection.

    PubMed

    Ott, Lisa; Scholz, Brigitte; Höller, Martina; Hasselt, Kristin; Ensser, Armin; Burkovski, Andreas

    2013-01-01

    Corynebacterium diphtheriae, the causative agent of diphtheria, has been thoroughly studied with respect to toxin production and pili formation, while knowledge on host responses to C. diphtheriae infection is limited. In this study, we studied adhesion to and invasion of epithelial cells by different C. diphtheriae isolates. When NFκ-B reporter cell lines were used to monitor the effect of C. diphtheriae infection on human cells, strain-specific differences were observed. While adhesion to host cells had no effect, a correlation of invasion rate with NFκ-B induction was found, which indicates that internalization of bacteria is crucial for NFκ-B induction. Immunofluorescence microscopy experiments used to support the reporter assays showed that translocation of p65, as a hallmark of NFκ-B induction, was only observed in association with cell invasion by C. diphtheriae. Our data indicate that the response of epithelial cells to C. diphtheriae infection is determined by internalization of bacteria and that invasion of these cells is an active process; tetracycline-treated C. diphtheriae was still able to attach to host cells, but lost its ability to invade the cytoplasm. Recognition of pathogen-associated molecular patterns such as pili subunits by membrane-bound receptors facing the outside of the cell is not sufficient for NFκ-B induction.

  7. Toxigenic Corynebacterium ulcerans in human and non-toxigenic Corynebacterium diphtheriae in cat.

    PubMed

    Detemmerman, L; Rousseaux, D; Efstratiou, A; Schirvel, C; Emmerechts, K; Wybo, I; Soetens, O; Piérard, D

    2013-10-01

    Corynebacterium diphtheriae and Corynebacterium ulcerans are rarely isolated from clinical samples in Belgium. A case of toxigenic C. ulcerans in a woman is described, which confirms that this pathogen is still present. During investigation of the patient's cats, only a non-toxigenic toxin-bearing C. diphtheriae strain was detected.

  8. Characterization of Corynebacterium diphtheriae isolates from infected skin lesions in the Northern Territory of Australia.

    PubMed

    Gordon, Claire L; Fagan, Peter; Hennessy, Jann; Baird, Robert

    2011-11-01

    Corynebacterium diphtheriae is commonly isolated from cutaneous skin lesions in the Northern Territory of Australia. We prospectively assessed 32 recent isolates from infected skin lesions, in addition to reviewing 192 isolates collected over 5 years for toxin status. No isolates carried the toxin gene. Toxigenic C. diphtheriae is now a rare occurrence in the Northern Territory.

  9. Characterization and comparison of invasive Corynebacterium diphtheriae isolates from France and Poland.

    PubMed

    Farfour, E; Badell, E; Zasada, A; Hotzel, H; Tomaso, H; Guillot, S; Guiso, N

    2012-01-01

    Corynebacterium diphtheriae, the agent of diphtheria, is rarely responsible for bacteremia. However, high numbers of bacteremia have been reported in countries with extensive immunization coverage. Here, we used molecular and phenotypic tools to characterize and compare 42 invasive isolates collected in France (including New Caledonia) and Poland over a 23-year period.

  10. First Report on the Draft Genome Sequences of Corynebacterium diphtheriae Isolates from India

    PubMed Central

    Anandan, Shalini; Rajamani Sekar, Suresh Kumar; Gopi, Radha; Devanga Ragupathi, Naveen Kumar; Ramesh, Srilekha; Verghese, Valsan Philip; Korulla, Sophy; Mathai, Sarah; Sangal, Lucky; Joshi, Sudhir

    2016-01-01

    We report here the draft genome sequences of five Corynebacterium diphtheriae isolates of Indian origin. The C. diphtheriae isolates TH1141, TH510, TH1526, TH1337, and TH2031 belong to sequence type ST-50, ST-295, ST-377, ST-405, and ST-405, with an average genome size of 2.5 Mbp. PMID:27881543

  11. An asymmetric and slightly dimerized structure for the tetanus toxoid protein used in glycoconjugate vaccines.

    PubMed

    Abdelhameed, Ali Saber; Morris, Gordon A; Adams, Gary G; Rowe, Arthur J; Laloux, Olivier; Cerny, Louis; Bonnier, Benjamin; Duvivier, Pierre; Conrath, Karel; Lenfant, Christophe; Harding, Stephen E

    2012-11-06

    Tetanus toxoid protein has been characterized with regard oligomeric state and hydrodynamic (low-resolution) shape, important parameters with regard its use in glycoconjugate vaccines. From sedimentation velocity and sedimentation equilibrium analysis in the analytical ultracentrifuge tetanus toxoid protein is shown to be mostly monomeric in solution (~86%) with approximately 14% dimer. The relative proportions do not appear to change significantly with concentration, suggesting the two components are not in reversible equilibrium. Hydrodynamic solution conformation studies based on high precision viscometry, combined with sedimentation data show the protein to be slightly extended conformation in solution with an aspect ratio ~3. The asymmetric structure presents a greater surface area for conjugation with polysaccharide than a more globular structure, underpinning its popular choice as a conjugation protein for glycoconjugate vaccines.

  12. A Nonadjuvanted Transcutaneous Tetanus Patch Is Effective in Boosting Anti-Tetanus Toxoid Immune Responses

    PubMed Central

    Seid, Robert C.; Reinisch, Christoph; Schlegl, Robert; Moehlen, Michael; Meinke, Andreas

    2014-01-01

    Dry tetanus toxoid (TTx) patches were formulated without any adjuvant, with excipients to impart antigen stabilization and to enhance skin delivery. The booster effects of the TTx patches were assessed using a guinea pig model. The study revealed significant rises in TTx IgG titers induced by the TTx patches after a low-dose subcutaneous (s.c.) prime with TTx adsorbed to aluminum hydroxide. The TTx patch can therefore be considered an effective alternative to a subcutaneous booster. PMID:24334688

  13. Detection of anti-tetanus toxoid antibody on modified polyacrylonitrile fibers.

    PubMed

    Jain, Swati; Chattopadhyay, Sruti; Jackeray, Richa; Zainul Abid, C K V; Kumar, Manoj; Singh, Harpal

    2010-10-15

    Accurate determination of concentration of immunoglobulin (IgG) to tetanus toxoid is important in order to evaluate the immunogenicity of tetanus toxoid vaccines, immune competence in individual patients and to measure the prevalence of immunity in populations. Surface modified polyacrylonitrile (PAN) fibers were evaluated as a matrix to develop highly sensitive method for the detection of anti-tetanus antibody in a sandwich ELISA format. In the proposed method tetanus toxoid immobilized on modified PAN fibers was used to detect anti-tetanus antibody (raised in horse hence represented as horse anti-tetanus toxoid or HAT-Ab) with horse raddish peroxidase enzyme conjugated with Rabbit anti-Horse IgG (RAH-HRP) as the label within 2.5h. A sigmoidal pattern for the detection of different concentration of antibody ranging from 1.0 to 0.0001 IU mL(-1) was validated. The immunoassay recorded a very high sensitivity as concentration as low as 0.0005 IU mL(-1) of HAT-Ab was detected. The intra- and inter-assay precision for 3 parallel measurements of 0.01 and for 0.001 IU mL(-1) of antibody varied from 5.4% to 11% and 5.7% to 20% respectively. PAN fibers were also used to qualitatively access the presence of different level of anti-tetanus antibody spiked in human blood. Seroepidemiological studies to measure the immunity against tetanus were conducted with twenty-five human beings belonging to various age groups using modified PAN-ELISA. The sensitivity, specificity and the reproducibility of the developed immunoassay indicate the potential application of modified PAN fibers in the field of immunodiagnostics.

  14. A nonadjuvanted transcutaneous tetanus patch is effective in boosting anti-tetanus toxoid immune responses.

    PubMed

    Seid, Robert C; Reinisch, Christoph; Schlegl, Robert; Moehlen, Michael; Meinke, Andreas; Lundberg, Urban

    2014-02-01

    Dry tetanus toxoid (TTx) patches were formulated without any adjuvant, with excipients to impart antigen stabilization and to enhance skin delivery. The booster effects of the TTx patches were assessed using a guinea pig model. The study revealed significant rises in TTx IgG titers induced by the TTx patches after a low-dose subcutaneous (s.c.) prime with TTx adsorbed to aluminum hydroxide. The TTx patch can therefore be considered an effective alternative to a subcutaneous booster.

  15. Rapid detection and molecular differentiation of toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans strains by LightCycler PCR.

    PubMed

    Sing, Andreas; Berger, Anja; Schneider-Brachert, Wulf; Holzmann, Thomas; Reischl, Udo

    2011-07-01

    The systemic symptoms of diphtheria are caused by the tox-encoded diphtheria toxin (DT) which is produced by toxigenic Corynebacterium spp. Besides the classical agent C. diphtheriae, the zoonotic pathogen C. ulcerans has increasingly been reported as an emerging pathogen for diphtheria. The reliable detection of toxigenic Corynebacterium spp. is of substantial importance for both diphtheria surveillance in the public health sector and the clinical workup of a patient with diphtherialike symptoms. Since the respective tox genes of C. diphtheriae and C. ulcerans differ from each other in both DNA and amino acid sequence, both tox genes should be covered by novel real-time PCR methods. We describe the development and validation of a LightCycler PCR assay which reliably recognizes tox genes from both C. diphtheriae and C. ulcerans and differentiates the respective target genes by fluorescence resonance energy transfer (FRET) hybridization probe melting curve analysis.

  16. Toxoids of Pseudomonas aeruginosa exotoxin-A: photoaffinity inactivation of purified toxin and purified toxin derivatives.

    PubMed Central

    Callahan, L T; Martinez, D; Marburg, S; Tolman, R L; Galloway, D R

    1984-01-01

    For the preparation of greatly detoxified but highly immunogenic toxoids, two enzymatically active, low-toxicity derivatives of Pseudomonas aeruginosa exotoxin-A were further inactivated by photoaffinity labeling. These derivatives were formed during toxin purification, when a relatively crude toxin preparation was concentrated by ammonium sulfate precipitation and subsequently dialyzed. These derivatives, designated peak-1 protein (PK-1) and peak-2 protein (PK-2) were antigenically indistinguishable from native toxin, but had isoelectric points (5.00 and 4.90, respectively) that were different from that of the native toxin (4.95). Although the enzymatic activities and molecular weights of PK-1 and PK-2 were similar to those of native toxin, their toxicities were greatly reduced (ca. 500-fold). Photoaffinity labeling of fully active toxin-A, purified by a process which limits the formation of these derivatives, decreased its enzymatic activity (ca. 30-fold) and toxicity (ca. 100-fold). Likewise, photoaffinity labeling of purified PK-1 and PK-2 decreased their enzymatic activities and toxicities (ca. 30-fold and 100-fold, respectively) and, thus, yielded toxoids that were ca. 50,000-fold less toxic than unpurified native toxin. These toxoids were irreversibly detoxified and highly immunogenic during 9 months of storage at 4 degrees C. Images PMID:6321348

  17. Comparative Immunogenicity of the Tetanus Toxoid and Recombinant Tetanus Vaccines in Mice, Rats, and Cynomolgus Monkeys

    PubMed Central

    Yu, Rui; Fang, Ting; Liu, Shuling; Song, Xiaohong; Yu, Changming; Li, Jianmin; Fu, Ling; Hou, Lihua; Xu, Junjie; Chen, Wei

    2016-01-01

    Tetanus is caused by the tetanus neurotoxin (TeNT) and is one of the most dreaded diseases especially in the developing countries. The current vaccine against tetanus is based on an inactivated tetanus toxin, which is effective but has many drawbacks. In our previous study, we developed a recombinant tetanus vaccine based on protein TeNT-Hc, with clear advantages over the toxoid vaccine in terms of production, characterization, and homogeneity. In this study, the titers, growth extinction, and persistence of specific antibodies induced by the two types of vaccine in mice, rats, and cynomolgus monkeys were compared. The booster vaccination efficacy of the two types of vaccines at different time points and protection mechanism in animals were also compared. The recombinant tetanus vaccine induced persistent and better antibody titers and strengthened the immunity compared with the commercially available toxoid vaccine in animals. Our results provide a theoretical basis for the development of a safe and effective recombinant tetanus vaccine to enhance the immunity of adolescents and adults as a substitute for the current toxoid vaccine. PMID:27348002

  18. Comparative Immunogenicity of the Tetanus Toxoid and Recombinant Tetanus Vaccines in Mice, Rats, and Cynomolgus Monkeys.

    PubMed

    Yu, Rui; Fang, Ting; Liu, Shuling; Song, Xiaohong; Yu, Changming; Li, Jianmin; Fu, Ling; Hou, Lihua; Xu, Junjie; Chen, Wei

    2016-06-25

    Tetanus is caused by the tetanus neurotoxin (TeNT) and is one of the most dreaded diseases especially in the developing countries. The current vaccine against tetanus is based on an inactivated tetanus toxin, which is effective but has many drawbacks. In our previous study, we developed a recombinant tetanus vaccine based on protein TeNT-Hc, with clear advantages over the toxoid vaccine in terms of production, characterization, and homogeneity. In this study, the titers, growth extinction, and persistence of specific antibodies induced by the two types of vaccine in mice, rats, and cynomolgus monkeys were compared. The booster vaccination efficacy of the two types of vaccines at different time points and protection mechanism in animals were also compared. The recombinant tetanus vaccine induced persistent and better antibody titers and strengthened the immunity compared with the commercially available toxoid vaccine in animals. Our results provide a theoretical basis for the development of a safe and effective recombinant tetanus vaccine to enhance the immunity of adolescents and adults as a substitute for the current toxoid vaccine.

  19. Microbiological changes and diversity in autochthonous non-toxigenic Corynebacterium diphtheriae isolated in France.

    PubMed

    Farfour, E; Badell, E; Dinu, S; Guillot, S; Guiso, N

    2013-10-01

    Autochtonous toxigenic Corynebacterium diphtheriae have disappeared in mainland France, but non-toxigenic C. diphtheriae are still circulating. Using phenotypic and molecular tools, we retrospectively characterized 103 non-toxigenic C. diphtheriae collected in mainland France and highlight several changes. The proportion of C. diphtheriae belfanti increased between 1977 and 2011 and it is the most frequent biotype recovered in recent years. Resistance to ciprofloxacin has increased and most isolates with decreased sensitivity belong to the belfanti biotype. Using multilocus sequence typing, we demonstrate that French isolates are distributed in a large number of sequence types and identify three distinct lineages. C. diphtheriae mitis and gravis form lineage I while C. diphtheriae belfanti forms lineages II and III. Almost all isolates of lineage II are part of a unique clonal complex or are very close to it. Most French isolates have a dtxR sequence homologous to that of toxigenic isolates, suggesting that if lyzogenised by a corynephage, they can express diphtheria toxin.

  20. Structure of a DsbF homologue from Corynebacterium diphtheriae.

    PubMed

    Um, Si-Hyeon; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

    2014-09-01

    Disulfide-bond formation, mediated by the Dsb family of proteins, is important in the correct folding of secreted or extracellular proteins in bacteria. In Gram-negative bacteria, disulfide bonds are introduced into the folding proteins in the periplasm by DsbA. DsbE from Escherichia coli has been implicated in the reduction of disulfide bonds in the maturation of cytochrome c. The Gram-positive bacterium Mycobacterium tuberculosis encodes DsbE and its homologue DsbF, the structures of which have been determined. However, the two mycobacterial proteins are able to oxidatively fold a protein in vitro, unlike DsbE from E. coli. In this study, the crystal structure of a DsbE or DsbF homologue protein from Corynebacterium diphtheriae has been determined, which revealed a thioredoxin-like domain with a typical CXXC active site. Structural comparison with M. tuberculosis DsbF would help in understanding the function of the C. diphtheriae protein.

  1. Structure of a DsbF homologue from Corynebacterium diphtheriae

    PubMed Central

    Um, Si-Hyeon; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

    2014-01-01

    Disulfide-bond formation, mediated by the Dsb family of proteins, is important in the correct folding of secreted or extracellular proteins in bacteria. In Gram-negative bacteria, disulfide bonds are introduced into the folding proteins in the periplasm by DsbA. DsbE from Escherichia coli has been implicated in the reduction of disulfide bonds in the maturation of cytochrome c. The Gram-positive bacterium Mycobacterium tuberculosis encodes DsbE and its homologue DsbF, the structures of which have been determined. However, the two mycobacterial proteins are able to oxidatively fold a protein in vitro, unlike DsbE from E. coli. In this study, the crystal structure of a DsbE or DsbF homologue protein from Corynebacterium diphtheriae has been determined, which revealed a thioredoxin-like domain with a typical CXXC active site. Structural comparison with M. tuberculosis DsbF would help in understanding the function of the C. diphtheriae protein. PMID:25195886

  2. Production of inflammatory cytokines in response to diphtheria-pertussis-tetanus (DPT), haemophilus influenzae type b (Hib), and 7-valent pneumococcal (PCV7) vaccines

    PubMed Central

    Kashiwagi, Yasuyo; Miyata, Akiko; Kumagai, Takuji; Maehara, Kouji; Suzuki, Eitarou; Nagai, Takao; Ozaki, Takao; Nishimura, Naoko; Okada, Kenji; Kawashima, Hisashi; Nakayama, Tetsuo

    2014-01-01

    Haemophilus influenzae type b (Hib) and 7-valent pneumococcal (PCV7) vaccines both became recommended in Japan in 2010. In this study, cytokine production was investigated in peripheral blood mononuclear cells (PBMCs) cultures stimulated with diphtheria and tetanus toxoids combined with acellular pertussis vaccine (DPT), Hib, and PCV7 separately or concurrent different combinations, all as final off-the-shelf vaccines without the individual vaccine components as controls. Higher IL-1β levels were produced when cultures were stimulated with PCV than with DPT or Hib, and the concurrent stimulation including PCV7 enhanced the production of IL-1β. Although Hib induced higher levels of IL-6, no significant difference was observed in IL-6 production with the concurrent stimulation. The concurrent stimulation with Hib/PCV7 and DPT/Hib/PCV7 produced higher levels of TNF-α and human G-CSF. Cytokine profiles were examined in serum samples obtained from 61 vaccine recipients with febrile reactions and 18 recipients without febrile illness within 24 h of vaccination. No significant difference was observed in cytokine levels of IL-1β, IL-4, IL-6, IL-10, IL-12, IFN-γ, MIP-1, TNF-α, and prostaglandin E2 (PGE2) in sera between the two groups. However, significantly higher levels of human G-CSF were observed in recipients with febrile illness than in those without febrile reactions. Further investigations of the significance of elevated serum G-CSF levels are required in vaccine recipients with febrile illness. PMID:24589970

  3. [Adhesion of corynebacterium diphtheriae: the role of surface structures and formation mechanism].

    PubMed

    Kharseeva, G G; Alieva, A A

    2014-01-01

    The paper is devoted to the study of surface structures including pili (fimbriae) 67-72p surface protein, DIP 1281 surface protein, lipoarabinomannan CdiLAM and their role in the adhesion and colonization of the mucous membrane of the throat by Corynebacterium diphtheriae. A description is offered for the main stages in the adhesion process of diphtheria causative agent and the ability of its adhesins to stimulate the effect of innate and acquired immunity factors. The paper stresses prospectiveness of the development of vaccines forming immunoprotection of the organism against adhesive activity of C. diphtheriae and also preventing their colonization and reproduction. That would facilitate a solution for the problem of diphtheria carrier state, which cannot be solved using the existing means of preventive vaccination.

  4. Tetanus and diphtheria antibodies and response to a booster dose in Brazilian HIV-1-infected women.

    PubMed

    Bonetti, Tatiana C S; Succi, Regina C M; Weckx, Lily Y; Tavares-Lopes, L; de Moraes-Pinto, M Isabel

    2004-09-09

    Tetanus and diphtheria (Td) antibodies were studied in HIV-1-infected women during puerperium. HIV group (n=61) was compared with Control group (n=101). Twenty-one women from HIV and 13 from Control group who had antibody levels lower than 0.1 IU/mL received a booster with Td vaccine. Antibodies were assessed by double antigen ELISA. Mean tetanus and diphtheria antibody levels from HIV group were lower than those from Control group. Multiple linear regression analysis showed that tetanus and diphtheria antibody levels were decreased by HIV-1-infection, and that was independent of the reduction due to the time interval between last booster and antibody assessment. After a booster dose, both groups had an increase in mean tetanus and diphtheria antibody levels, but in Control group the levels were higher than in HIV group.

  5. Diphtheria and the Vaccine (Shot) to Prevent It: Information for Parents

    MedlinePlus

    ... serious disease (and also protects against tetanus and whooping cough) • Prevents your child from developing a thick coating ... is effective at preventing diphtheria (as well as whooping cough and tetanus). Vaccines, like any medicine, can have ...

  6. The problem of the periodicity of the epidemic process. [solar activity effects on diphtheria outbreak

    NASA Technical Reports Server (NTRS)

    Yagodinskiy, V. N.; Konovalenko, Z. P.; Druzhinin, I. P.

    1974-01-01

    An analysis of data from epidemics makes it possible to determine their principal causes, governed by environmental factors (solar activity, etc.) The results of an analysis of the periodicity of the epidemic process in the case of diphtheria are presented which was conducted with the aid of autocorrelation and spectral methods of analysis. Numerical data (annual figures) are used on the dynamics of diphtheria in 50 regions (points) with a total duration of 2,777 years.

  7. Colonisation with toxigenic Corynebacterium diphtheriae in a Scottish burns patient, June 2015.

    PubMed

    Deshpande, Ashutosh; Inkster, Teresa; Hamilton, Kate; Litt, David; Fry, Norman; Kennedy, Iain T R; Shookhye-Dickson, Jacqueline; Hill, Robert L R

    2015-01-01

    On 12 June 2015, Corynebacterium diphtheriae was identified in a skin swab from a burns patient in Scotland. The isolate was confirmed to be genotypically and phenotypically toxigenic. Multilocus sequence typing of three patient isolates yielded sequence type ST 125. The patient was clinically well. We summarise findings of this case, and results of close contact identification and screening: 12 family and close contacts and 32 hospital staff have been found negative for C. diphtheriae.

  8. Potential pathogenic role of aggregative-adhering Corynebacterium diphtheriae of different clonal groups in endocarditis.

    PubMed

    Hirata Jr, R; Pereira, G A; Filardy, A A; Gomes, D L R; Damasco, P V; Rosa, A C P; Nagao, P E; Pimenta, F P; Mattos-Guaraldi, A L

    2008-11-01

    Invasive diseases caused by Corynebacterium diphtheriae have been described increasingly. Several reports indicate the destructive feature of endocarditis attributable to nontoxigenic strains. However, few reports have dealt with the pathogenicity of invasive strains. The present investigation demonstrates a phenotypic trait that may be used to identify potentially invasive strains. The study also draws attention to clinical and microbiological aspects observed in 5 cases of endocarditis due to C. diphtheriae that occurred outside Europe. Four cases occurred in female school-age children (7-14 years) treated at different hospitals in Rio de Janeiro, Brazil. All patients developed other complications including septicemia, renal failure and/or arthritis. Surgical treatment was performed on 2 patients for valve replacement. Lethality was observed in 40% of the cases. Microorganisms isolated from 5 blood samples and identified as C. diphtheriae subsp mitis (N = 4) and C. diphtheriae subsp gravis (N = 1) displayed an aggregative adherence pattern to HEp-2 cells and identical one-dimensional SDS-PAGE protein profiles. Aggregative-adhering invasive strains of C. diphtheriae showed 5 distinct RAPD profiles. Despite the clonal diversity, all 5 C. diphtheriae invasive isolates seemed to display special bacterial adhesive properties that may favor blood-barrier disruption and systemic dissemination of bacteria. In conclusion, blood isolates from patients with endocarditis exhibited a unique adhering pattern, suggesting a pathogenic role of aggregative-adhering C. diphtheriae of different clones in endocarditis. Accordingly, the aggregative-adherence pattern may be used as an indication of some invasive potential of C. diphtheriae strains.

  9. Tetanus and diphtheria immunity in adolescents from São Paulo, Brazil.

    PubMed

    Dinelli, M I S; Fisberg, M; de Moraes-Pinto, M I

    2007-02-01

    Tetanus and diphtheria vaccines are of special concern in adolescents because boosters are necessary for adequate maintenance of protection and are often omitted. We assessed serum levels of tetanus and diphtheria antibodies in adolescents and their association with vaccination status. From May to October 2001, we evaluated the vaccination records of 208 adolescents aged 10 to 20 years in São Paulo, Brazil. Antibodies to tetanus and diphtheria were detected using double-antigen ELISA and vaccination records were analyzed according to the guidelines of the Brazilian National Immunization Program. All adolescents had received complete primary vaccinations against tetanus and diphtheria, but 23.1% of them had not received a booster dose in the last 10 years. All adolescents were immune to tetanus and 88.9% were fully protected (antibodies (3)0.1 IU/mL). One individual (0.5%) was non-immune to diphtheria and 86% were fully protected against the disease. Adolescents with up-to-date vaccination records had higher antibody levels than those with not up-to-date records for tetanus (0.763 vs 0.239 IU/mL, t-test: P < 0.0001) and diphtheria (0.366 vs 0.233 IU/mL, t-test: P = 0.014). Full immunity against tetanus (antibodies (3)0.1 IU/mL) was higher among individuals with up-to-date vaccination (93.1%) when compared to those with not up-to-date records (75%, Fisher's exact test: P = 0.001). All adolescents had received basic immunization in childhood and were protected against tetanus and diphtheria. However, these data indicate that more emphasis should be placed on the tetanus-diphtheria booster in order to avoid a decay in antibody levels.

  10. Structural correlates of carrier protein recognition in tetanus toxoid-conjugated bacterial polysaccharide vaccines

    PubMed Central

    Lockyer, Kay; Gao, Fang; Derrick, Jeremy P.; Bolgiano, Barbara

    2015-01-01

    An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the HC domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8 × 106 g/mol to larger than 20 × 106 g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the HC domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the HC domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines. PMID:25640334

  11. Structural correlates of carrier protein recognition in tetanus toxoid-conjugated bacterial polysaccharide vaccines.

    PubMed

    Lockyer, Kay; Gao, Fang; Derrick, Jeremy P; Bolgiano, Barbara

    2015-03-10

    An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the HC domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8×10(6) g/mol to larger than 20×10(6) g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the HC domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the HC domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines.

  12. Characterization of DIP0733, a multi-functional virulence factor of Corynebacterium diphtheriae.

    PubMed

    Antunes, Camila Azevedo; Sanches dos Santos, Louisy; Hacker, Elena; Köhler, Stefanie; Bösl, Korbinian; Ott, Lisa; de Luna, Maria das Graças; Hirata, Raphael; Azevedo, Vasco Ariston de Carvalho; Mattos-Guaraldi, Ana-Luíza; Burkovski, Andreas

    2015-03-01

    Corynebacterium diphtheriae is typically recognized as an extracellular pathogen. However, a number of studies revealed its ability to invade epithelial cells, indicating a more complex pathogen-host interaction. The molecular mechanisms controlling and facilitating internalization of Cor. diphtheriae are poorly understood. In this study, we investigated the role of DIP0733 as virulence factor to elucidate how it contributes to the process of pathogen-host cell interaction. Based on in vitro experiments, it was suggested recently that the DIP0733 protein might be involved in adhesion, invasion of epithelial cells and induction of apoptosis. A corresponding Cor. diphtheriae mutant strain generated in this study was attenuated in its ability to colonize and kill the host in a Caenorhabditis elegans infection model system. Furthermore, the mutant showed an altered adhesion pattern and a drastically reduced ability to adhere and invade epithelial cells. Subsequent experiments showed an influence of DIP0733 on binding of Cor. diphtheriae to extracellular matrix proteins such as collagen and fibronectin. Furthermore, based on its fibrinogen-binding activity, DIP0733 may play a role in avoiding recognition of Cor. diphtheriae by the immune system. In summary, our findings support the idea that DIP0733 is a multi-functional virulence factor of Cor. diphtheriae.

  13. Serological survey on the immunity to diphtheria of the northern Greek population.

    PubMed

    Souliou, E; Kyriazopoulou, V; Diza, E; Hatzistylianou, M; Frantzidou, F

    1997-07-01

    The recent outbreak of diphtheria in the Newly Independent States (NIS) of the former USSR and the immigration from these high risk areas to Greece prompted us to determine the diphtheria antitoxin levels by enzyme-linked immunosorbent assays (ELISA) in 509 healthy individuals (307 males and 202 females) from northern Greece. The population under study was divided in ten age groups from 1 day to > 60 years old. Diphtheria antitoxin levels of > or = 0.1 IU/ml were considered as protective ones. 44.6% of the examined people were found susceptible. The children up to their twenties seem to be immune to diphtheria in a high proportion (86-88.4%). The diphtheria antitoxin levels declined sharply above this age (17.6% in the age group 21-30 years old). The level of protection in adults appeared to be higher in the oldest group (49%). According to these results, the adults are not properly protected. Booster doses of vaccine for them are recommended to improve the resistance of the northern Greek population from possible infection by toxigenic stains of Corynebacterium diphtheriae, imported or endogenous.

  14. Multilocus sequence typing identifies evidence for recombination and two distinct lineages of Corynebacterium diphtheriae.

    PubMed

    Bolt, Frances; Cassiday, Pamela; Tondella, Maria Lucia; Dezoysa, Aruni; Efstratiou, Androulla; Sing, Andreas; Zasada, Aleksandra; Bernard, Kathryn; Guiso, Nicole; Badell, Edgar; Rosso, Marie-Laure; Baldwin, Adam; Dowson, Christopher

    2010-11-01

    We describe the development of a multilocus sequence typing (MLST) scheme for Corynebacterium diphtheriae, the causative agent of the potentially fatal upper respiratory disease diphtheria. Global changes in diphtheria epidemiology are highlighted by the recent epidemic in the former Soviet Union (FSU) and also by the emergence of nontoxigenic strains causing atypical disease. Although numerous techniques have been developed to characterize C. diphtheriae, their use is hindered by limited portability and, in some instances, poor reproducibility. One hundred fifty isolates from 18 countries and encompassing a period of 50 years were analyzed by multilocus sequence typing (MLST). Strain discrimination was in accordance with previous ribotyping data, and clonal complexes associated with disease outbreaks were clearly identified by MLST. The data produced are portable, reproducible, and unambiguous. The MLST scheme described provides a valuable tool for monitoring and characterizing endemic and epidemic C. diphtheriae strains. Furthermore, multilocus sequence analysis of the nucleotide data reveals two distinct lineages within the population of C. diphtheriae examined, one of which is composed exclusively of biotype belfanti isolates and the other of multiple biotypes.

  15. A lack of genetic basis for biovar differentiation in clinically important Corynebacterium diphtheriae from whole genome sequencing.

    PubMed

    Sangal, Vartul; Burkovski, Andreas; Hunt, Alison C; Edwards, Becky; Blom, Jochen; Hoskisson, Paul A

    2014-01-01

    The differentiation of clinically important Corynebacterium diphtheriae into specific biovars is complex and phylogenetically unclear. Comparative genomic analyses of 17 strains indicate that the division of C. diphtheriae into different biovars does not correlate with the variation in the gene content in the relevant metabolic categories that are potentially involved in the biovar discrimination. The biochemical separation is also not supported by phylogenetic analyses, suggesting molecular methods of typing C. diphtheriae strains should be adopted much more widely.

  16. Effects of simultaneous immunization of Haemophilus influenzae type b conjugate vaccine and diphtheria-tetanus-acellular pertussis vaccine on anti-tetanus potencies in mice, guinea pigs, and rats.

    PubMed

    Fukuda, Tadashi; Iwaki, Masaaki; Komiya, Takako; Shibayama, Keigo; Takahashi, Motohide; Nakashima, Hideki

    2013-01-01

    Haemophilus influenzae type b vaccine conjugated with tetanus toxoid (HibT) was licensed for use in childhood immunization in Japan in 2007. As adsorbed diphtheria-tetanus-acellular pertussis (DTaP) combined with HibT vaccine has not been introduced in Japan, DTaP and HibT vaccines are injected at separate sites with a similar immunization schedule. There are various interfering or stimulatory effects between components of combined vaccines contained in DTaP and HibT vaccines. In this study, we investigated the effect of HibT containing combination vaccines on anti-tetanus potencies by using animal models (mouse, guinea pig, and rat). HibT vaccine and HibT components of imported DTaP-HibT vaccine alone showed comparable or higher anti-tetanus potency than DTaP vaccine and DTaP-containing components of combination vaccines. Mixing these components before injection resulted in potencies greater than the sum of individual potencies. Injecting individual components at separate sites in animals resulted in potency roughly equivalent to the sum of the individual potencies. These results provide useful information regarding the use of HibT-containing multivalent vaccines in childhood immunization.

  17. Laboratory guidelines for the diagnosis of infections caused by Corynebacterium diphtheriae and C. ulcerans. World Health Organization.

    PubMed

    Efstratiou, A; George, R C

    1999-12-01

    These guidelines represent an application of the World Health Organization European Region's manual for the laboratory diagnosis of diphtheria for laboratories in the United Kingdom (UK), but they could be applied to laboratories overseas. The manual was rewritten in response to the re-emergence of diphtheria in eastern Europe and the emergence of other infections caused by Corynebacterium diphtheriae and C. ulcerans in the UK and overseas. The guidelines summarise our current recommendations and procedures for the microbiological diagnosis of infections caused by toxigenic and non-toxigenic isolates of corynebacteria, with particular reference to C. diphtheriae and C. ulcerans.

  18. Direct polymerase chain reaction for detection of toxigenic Corynebacterium diphtheriae strains from the Republic of Georgia after prolonged storage.

    PubMed

    Kobaidze, K; Popovic, T; Nakao, H; Quick, L

    2000-02-01

    A total of 226 paired nose and throat swab specimens from 113 clinical diphtheria cases from the republic of Georgia were analyzed by direct polymerase chain reaction targeting both A and B subunits of the diphtheria toxin gene, tox. Even after prolonged transport and extensive storage (7-14 months) of the clinical specimens in silica gel packages, direct polymerase chain reaction detected the diphtheria tox gene in 54% of the specimens. Specimens obtained by throat swab were three times more likely than those obtained by nose swab to be positive for Corynebacterium diphtheriae.

  19. The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine.

    PubMed

    Springer, S; Selbitz, H J

    1999-07-01

    Necrotic enteritis in sucking piglets constitutes a serious problem in piglet rearing units because of the high morbidity and mortality associated with the disease. The primary causal agent is Clostridium perfringens type C. The beta-toxin plays a decisive role in the pathogenesis of this disease. A toxoid vaccine for use in sows has been developed and studied in field trials. The European Pharmacopoeia Monograph on vaccines for use in animals lays down a method of the efficacy testing based on the immunization of rabbits, the collection of pooled sera and the subsequent assay of anti-toxin antibodies in mice using an appropriate test toxin. The vaccine is regarded as effective if it induces a minimum of 10 IU of beta-anti-toxin per ml of rabbit serum. We have established a range of 17.14-98.23 IU beta-anti-toxin per ml rabbit serum induced by a sample of C. perfringens toxoid vaccine. The vaccine has been used under field conditions in different rearing units at the same time, mostly in the form of emergency vaccinations following the outbreak of disease. The outcome of vaccination was evaluated by recording the total numbers of piglets born alive and the piglet losses. Use of the vaccine, coupled with other measures, resulted in an approximately 30% reduction in the number of losses.

  20. Cattle immune response to botulinum type D toxoid: results of a vaccination study.

    PubMed

    Steinman, A; Galon, N; Arazi, A; Bar-Giora, Y; Shpigel, N Y

    2007-11-01

    Cattle botulism is a food-borne intoxication caused by the ingestion of preformed botulinum neurotoxins (BoNT) of serotypes B, C, or D. Protection in cattle against botulinum intoxication is based on the presence of specific serum neutralizing antibodies upon exposure. Outbreaks in vaccinated cattle have raised concerns about vaccine quality and efficacy. To this end, three different immunization protocols and the effect of maternal anti-BoNT/D antibodies, at the priming dose, were analyzed in 2-month-old dairy calves. Based on previously determined protective anti-BoNT/D antibody levels analyzed in field outbreaks, the immune response to type D toxoids was analyzed using an in-house ELISA system. Here we show that using the current vaccination strategy of using a priming dose in 2-month-old calves followed by booster doses after 4 weeks and annually thereafter, did not result in continuous protective levels of anti-BoNT/D antibodies. As a result of this vaccination protocol, only 15-31% of cattle in parities 1-3 were protected at the time of the annual booster. Vaccination study in calves indicated that adding a 6-month booster dose to the current protocol resulted in continuous protective levels of anti-BoNT/D antibodies well above the cut-off protective levels. The presence of maternally derived anti-BoNT/D antibodies did not interfere with the immune response to toxoids that can be administered to 2-month-old calves.

  1. Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage. lambda. immunoexpression library

    SciTech Connect

    Mullinax, R.L.; Gross, E.A.; Amberg, J.R.; Hogrefe, H.H.; Kubitz, M.M.; Greener, A.; Alting-Mees, M.; Ardourel, D.; Short, J.M.; Sorge, J.A. ); Hay, B.N.; Shopes, B. )

    1990-10-01

    The authors have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and {kappa} light-chain variable and constant region domains, were inserted into modified bacteriophase {lambda} expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2{percent} in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. They estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.

  2. Real time and accelerated stability studies of Tetanus toxoid manufactured in public sector facilities of Pakistan.

    PubMed

    Parveen, Ghazala; Hussain, Shahzad; Malik, Farnaz; Begum, Anwar; Mahmood, Sidra; Raza, Naeem

    2013-11-01

    Tetanus is an acute illness represented by comprehensive increased inflexibility and spastic spasms of skeletal muscles. The poor quality tetanus toxoid vaccine can raise the prevalence of neonatal tetanus. WHO has taken numerous steps to assist national regulatory authorities and vaccine manufacturers to ensure its quality and efficacy. It has formulated international principles for stability evaluation of each vaccine, which are available in the form of recommendations and guidelines. The aim of present study was to ensure the stability of tetanus vaccines produced by National Institute of Health, Islamabad, Pakistan by employing standardized methods to ensure constancy of tetanus toxoid at elevated temperature, if during storage/transportation cold chain may not be maintained in hot weather. A total of three batches filled during full-scale production were tested. All Stability studies determination were performed on final products stored at 2-8°C and elevated temperatures in conformance with the ICH Guideline of Stability Testing of Biological Products. These studies gave comparison between real time shelf-life stability and accelerated stability studies. The findings indicate long﷓term thermo stability and prove that this tetanus vaccine can remain efficient under setting of routine use when suggested measures for storage and handling are followed in true spirit.

  3. Collaborative study for the calibration of a replacement International Standard for Tetanus Toxoid Adsorbed.

    PubMed

    Tierney, Rob; Stickings, Paul; Hockley, Jason; Rigsby, Peter; Iwaki, Masaaki; Sesardic, Dorothea

    2011-11-01

    We present the results of a collaborative study for the establishment of a replacement International Standard (IS) for Tetanus Toxoid Adsorbed. Two candidate preparations were included in the study, one of which was established as the 4th IS for Tetanus Toxoid Adsorbed at the WHO Expert Committee on Biological Standardization meeting in October 2010. This preparation was found to have a unitage of 490 IU/ampoule, based on calibration in guinea pig challenge assays. Results from mouse challenge assays suggest that the relative performance of two candidate preparations may differ significantly between guinea pigs and mice. The authors note that the number of laboratories that performed guinea pig challenge assays, which are used to calibrate and assign IU, is much lower than in previous collaborative studies and this may have implications for calibration of replacement standards in the future. The issue of assigning separate units to the IS for guinea pig and mouse assays is discussed. The study also assessed performance of the replacement standard in serological assays which are used as alternative procedures to challenge assays for tetanus potency testing. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays.

  4. Antidiphtheria antibody responses in patients and carriers of Corynebacterium diphtheriae in the Arkhangelsk region of Russia.

    PubMed

    Danilova, Elena; Jenum, Pål A; Skogen, Vegard; Pilnikov, Valentin F; Sjursen, Haakon

    2006-06-01

    Diphtheria is under control in industrialized countries. However, single cases and outbreaks still occur and the disease is not completely understood. Forty-three individuals suspected of having diphtheria who were referred to the Infectious Disease Hospital of Arkhangelsk from December 1994 to March 1995 were included in this study. Fifteen patients were diagnosed as having diphtheria and received equine hyperimmune antidiphtheria toxin antiserum, and 28 were diagnosed as carriers, 12 with nondiphtherial tonsillitis or pharyngitis and 16 without symptoms. Serum samples were obtained on admission and during the course of the disease or during follow-up of carrier status. Samples were analyzed for antibodies against diphtheria toxin with both an in vitro neutralization test (NT) and a human-specific enzyme immunoassay. All of the cases but one were confirmed by a positive culture. Twelve patients had pharyngeal diphtheria, and three had combined laryngeal and pharyngeal disease. Half of the patients had life-threatening disease, and one died. On admission, the median antibody titers measured with the NT were 0.085 IU/ml for the patients, 5.12 IU/ml for the symptomatic carriers, and 10.24 IU/ml for the healthy carriers. All of the diphtheria patients but one and nine of the carriers (six symptomatic and three healthy) had increased antibody levels during the first 7 to 10 days after admission. No obvious correlation was revealed between the antibody level or its kinetics and the course of the disease. Antibody levels on admission of >1 IU/ml were associated with a low risk of diphtheria.

  5. [THE COMPARATIVE ANALYSIS OF TECHNIQUES OF IDENTIFICATION OF CORYNEBACTERIUM NON DIPHTHERIAE].

    PubMed

    Kharseeva, G G; Voronina, N A; Mironov, A Yu; Alutina, E L

    2015-12-01

    The comparative analysis was carried out concerning effectiveness of three techniques of identification of Corynebacterium non diphtheriae: bacteriological, molecular genetic (sequenation on 16SpRNA) andmass-spectrometric (MALDI-ToFMS). The analysis covered 49 strains of Corynebacterium non diphtheriae (C.pseudodiphheriticum, C.amycolatum, C.propinquum, C.falsenii) and 2 strains of Corynebacterium diphtheriae isolated under various pathology form urogenital tract and upper respiratory ways. The corinbacteria were identified using bacteriologic technique, sequenation on 16SpRNA and mass-spectrometric technique (MALDIToF MS). The full concordance of results of species' identification was marked in 26 (51%) of strains of Corynebacterium non diphtheriae at using three analysis techniques; in 43 (84.3%) strains--at comparison of bacteriologic technique with sequenation on 16S pRNA and in 29 (57%)--at mass-spectrometric analysis and sequenation on 16S pRNA. The bacteriologic technique is effective for identification of Corynebacterium diphtheriae. The precise establishment of species belonging of corynebacteria with variable biochemical characteristics the molecular genetic technique of analysis is to be applied. The mass-spectrometric technique (MALDI-ToF MS) requires further renewal of data bases for identifying larger spectrum of representatives of genus Corynebacterium.

  6. [The sensitivity to antibiotics of biofilm cultures of toxigenic strains Corynebacterium diphtheriae].

    PubMed

    Frolova, Ya N; Kharseyeva, G G; Mironov, A Yu

    2014-06-01

    The article presents analysis of sensitivity to antibacterial preparations of typical and biofilm culture of museum strain of Corynebacterium diphtheriae gravis tox+ SV-665. The strain was obtained from the L.A. Tarasevitch state research institute of standardization and control of medical biological preparations. The second strain C. diphtheriaecirculates gravis tox+ circulates in population of the Rostov oblast and it was recovered from patient with diagnosis of "localized form of diphtheria" by bacteriologic laboratory "1002 CGSEN SKVO" of Rostov-on-Don. The week and month biofilm cultures of both strains of C. diphtheriae gravis tox+ were used. The sensitivity to antibacterial preparations of typical and biofilm cultures of museum and circulating in population strains of agent of diphtheria were detected using minimal suppressing concentration by technique of serial dilutions in fluid growth medium. It is demonstrated that the most effective in respect of C. diphtheriae are such preparations as cefotaxinum, gentamycinum, lincomycin, canamycin and cefasolin. The sensitivity of pathogen in composition of biofilm to these preparations has no changes.

  7. Evaluation of human antibody responses to diphtheria toxin subunits A and B in various age groups.

    PubMed

    Karakus, R; Caglar, K; Aybay, C

    2007-11-01

    This study aimed to evaluate human antibody responses to diphtheria toxin subunits in various age groups. Antibodies against the intact diphtheria toxin and the diphtheria toxin subunits A and B were evaluated in 1319 individuals using a double-antigen ELISA. Although high levels of protection (83.6%, 95% CI 79.2-87.4) were found in children and adolescents, the middle-aged adult population was less protected (28.8%, 95% CI 24.3-33.6). An increase in age was associated with a decrease in the frequency of protected individuals in the 0-39-year age group (p <0.001). Anti-subunit B levels correlated well (p <0.01) with levels of antibodies against the intact toxin. In children aged < or =16 years, the intervals at which the peaks in geometric mean titres of anti-subunit B antibodies were observed were found to correlate with the ages at which booster doses are administered. Overall, males appeared to be more protected than females (OR 1.67, 95% CI 1.34-2.08, p <0.001). A small group of individuals had antibody levels of > or =0.1 IU/mL against the intact toxin, but did not have protective antibody against subunit B. Determination of anti-subunit B antibody levels should help in evaluating the effectiveness of diphtheria boosters and other aspects of diphtheria immunity.

  8. Prevalence of IgG diphtheria antitoxin in blood donors in Rio de Janeiro.

    PubMed

    Damasco, P V; Pimenta, F P; Filardy, A A; Brito, S M; Andrade, A F B; Lopes, G S; Hirata, R; Mattos-Guaraldi, A L

    2005-10-01

    The lack of information on the immunity of adults in Brazil against diphtheria prompted us to analyse sera from 234 blood donors aged 18-61 years (30.3% females and 69.7% males). IgG diphtheria antitoxin levels determined by means of an ELISA, validated by toxin neutralization test in Vero cells, showed that 30.7% (95% CI 25.0-37.1) of the population was fully protected (>or=1 IU/ml). The highest percentage of subjects fully protected was in the 31-40 years age group. Most of the subjects with uncertain or no protection (<1 IU/ml) were found in the 18-30 years age group (43.8%, OR 2.18, P=0.01). Antitoxin levels were not influenced by the increase in age. Males were more protected than females (80.5%, OR 0.44, P=0.01). The prevalence of 30% of individuals fully protected against diphtheria in blood donors in Rio de Janeiro supports the fact that immunity to diphtheria among healthy Brazilian adults is inadequate. To avoid diphtheria epidemics in the future the immunity among adults should be raised in the coming years.

  9. Calibration and commutability assessment of the 1st International Standard for Diphtheria Antitoxin Human.

    PubMed

    Stickings, Paul; Rigsby, Peter; Coombes, Laura; von Hunolstein, Christina; Ralli, Luisa; Pinto, Antonella; Sesardic, Dorothea

    2013-11-01

    The 1st International Standard for Diphtheria Antitoxin Human (coded 10/262) was established by the World Health Organization Expert Committee on Biological Standardization in 2012. This paper describes the production, characterization and calibration of the new standard which is intended for use in the standardization of assays used to measure diphtheria antibody responses in human serum. The new standard was calibrated in terms of the International Standard for Diphtheria Antitoxin Equine in an international collaborative study. A total of 8 participants from 8 different countries performed in vivo and/or in vitro toxin neutralization tests and returned data that was used to assign units to the proposed new standard. The new standard has a diphtheria antitoxin potency of 2 IU/ampoule and is predicted to be stable. A follow up study was performed to assess commutability of the new standard. The follow up study was an existing external quality assessment, modified to include the new standard. Results obtained suggest that the new standard is commutable, showing comparable behaviour to native human serum samples in the majority of the assays compared, and is therefore suitable for use as a reference preparation in assays used to measure the level of anti-diphtheria antibodies in human serum.

  10. Immunity to Diphtheria and Tetanus in Army Personnel and Adult Civilians in Mashhad, Iran.

    PubMed

    Hosseini Shokouh, Seyyed Javad; Mohammadi, Babak; Rajabi, Jalil; Mohammadian Roshan, Ghasem

    2017-03-24

    This study aimed to investigate serologic immunity to diphtheria and tetanus in army personnel and a sample population of adult civilians in Mashhad, Iran. Army personnel (n = 180) and civilians (n = 83) who presented at Mashhad army hospital participated in this study. Diphtheria and tetanus antitoxin levels were determined by enzyme-linked immunosorbent assay. Approximately 77% and 94% of army personnel aged 18-34 years had at least basic protection against diphtheria (antitoxin level ≥0.1 IU/mL) and tetanus (antitoxin level >0.1 IU/mL), respectively. For civilians in this age group, the proportions were 76% for both diseases. Antitoxin levels waned with age. Thus, participants older than 50 years had lower immunity; this decrease in immunity was more pronounced for tetanus than for diphtheria in both army personnel and civilians. For both diseases, geometric mean antitoxin titers and the proportion of participants with at least basic protection were higher in subjects with a history of vaccination in the last 10 years (P < 0.001), higher in men than women, and in army personnel than civilians in each age group. Young army personnel and civilians (18-34 years old) had adequate immunity to diphtheria and tetanus. However, the large number of susceptible older adults (>50 years old) calls for improved booster vaccination protocols.

  11. Diphtheria toxin treatment of Pet-1-Cre floxed diphtheria toxin receptor mice disrupts thermoregulation without affecting respiratory chemoreception

    PubMed Central

    Cerpa, Verónica; Gonzalez, Amalia; Richerson, George B.

    2014-01-01

    In genetically-modified Lmx1bf/f/p mice, selective deletion of LMX1B in Pet-1 expressing cells leads to failure of embryonic development of serotonin (5-HT) neurons. As adults, these mice have a decreased hypercapnic ventilatory response and abnormal thermoregulation. This mouse model has been valuable in defining the normal role of 5-HT neurons, but it is possible that developmental compensation reduces the severity of observed deficits. Here we studied mice genetically modified to express diphtheria toxin receptors (DTR) on Pet-1 expressing neurons (Pet-1-Cre/Floxed DTR or Pet1/DTR mice). These mice developed with a normal complement of 5-HT neurons. As adults, systemic treatment with 2 – 35 μg diphtheria toxin (DT) reduced the number of tryptophan hydroxylase immunoreactive (TpOH-ir) neurons in the raphe nuclei and ventrolateral medulla by 80%. There were no effects of DT on baseline ventilation (VE) or the ventilatory response to hypercapnia or hypoxia. At an ambient temperature (TA) of 24°C, all Pet1/DTR mice dropped their body temperature (TB) below 35°C after DT treatment, but the latency was shorter in males than females (3.0 ± 0.37 vs 4.57 ± 0.29 days, respectively; p < 0.001). One week after DT treatment, mice were challenged by dropping TA from 37°C to 24°C, which caused TB to decrease more in males than in females (29.7 ± 0.31°C vs 33.0 ± 1.3°C, p < 0.01). We conclude that the 20% of 5-HT neurons that remain after DT treatment in Pet1/DTR mice are sufficient to maintain normal baseline breathing and a normal response to CO2, while those affected include some essential for thermoregulation, in males more than females. In comparison to models with deficient embryonic development of 5-HT neurons, acute deletion of 5-HT neurons in adults leads to a greater defect in thermoregulation, suggesting that significant developmental compensation can occur. PMID:25171790

  12. Non-diphtheriae Corynebacterium species: an emerging respiratory pathogen.

    PubMed

    Díez-Aguilar, M; Ruiz-Garbajosa, P; Fernández-Olmos, A; Guisado, P; Del Campo, R; Quereda, C; Cantón, R; Meseguer, M A

    2013-06-01

    The purpose of the study was to describe the microbiological and clinical features of ten cases of lower respiratory tract infection due to Corynebacterium striatum, Corynebacterium propinquum and Corynebacterium pseudodiphtheriticum. Respiratory samples were recovered from hospitalised patients who were diagnosed of pneumonia and exacerbations of chronic respiratory infections. The samples were Gram-stained and seeded on conventional bacterial growing media. Bacteria were identified by matrix-assisted linear desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility was tested by the disk diffusion method. All patients presented an acute respiratory onset, most of them in the context of an underlying disease and/or immunosuppression. In all patients, the microscopical examination of Gram-stained respiratory samples showed numerous polymorphonuclear cells and Gram-positive bacilli, suggestive of the Corynebacterium morphotype. A pure culture growth of Corynebacterium was obtained in the majority (72 %) of samples. The conclusions are that non-diphtheriae Corynebacterium species are an emerging cause of respiratory infection among patients with chronic respiratory disease and/or immunosuppression, and cannot always be considered as mere colonisers. The microorganism's predominance in Gram-stained purulent respiratory samples together with abundant growth in the culture is the key for the microbiological diagnosis.

  13. An outbreak of diphtheria in a hospital for the mentally subnormal

    PubMed Central

    Anderson, G. S.; Penfold, J. B.

    1973-01-01

    An account is given of two separate outbreaks of diphtheria amongst mentally subnormal patients and nursing staff. In a total hospital population of about 1000 the number of people involved as carriers or cases was 60 and there were five deaths. The 60 people comprised 56 patients, of whom four were involved in both outbreaks, and four nurses. The organisms isolated were C. diphtheriae mitis but five strains were non-toxigenic. It is postulated that the outbreak began following the conversion of a non-toxigenic organism to a toxigenic one by bacteriophage action. The fatal cases were examples of membranous pharyngo-laryngo-tracheo-bronchial diphtheria with well marked pseudo-casts of the upper air passages. Images PMID:4200323

  14. Multilocus sequence types of invasive Corynebacterium diphtheriae isolated in the Rio de Janeiro urban area, Brazil.

    PubMed

    Viguetti, S Z; Pacheco, L G C; Santos, L S; Soares, S C; Bolt, F; Baldwin, A; Dowson, C G; Rosso, M L; Guiso, N; Miyoshi, A; Hirata, R; Mattos-Guaraldi, A L; Azevedo, V

    2012-04-01

    Invasive infections caused by Corynebacterium diphtheriae in vaccinated and non-vaccinated individuals have been reported increasingly. In this study we used multilocus sequence typing (MLST) to study genetic relationships between six invasive strains of this bacterium isolated solely in the urban area of Rio de Janeiro, Brazil, during a 10-year period. Of note, all the strains rendered negative results in PCR reactions for the tox gene, and four strains presented an atypical sucrose-fermenting ability. Five strains represented new sequence types. MLST results did not support the hypothesis that invasive (sucrose-positive) strains of C. diphtheriae are part of a single clonal complex. Instead, one of the main findings of the study was that such strains can be normally found in clonal complexes with strains related to non-invasive disease. Comparative analyses with C. diphtheriae isolated in different countries provided further information on the geographical circulation of some sequence types.

  15. [Etiologic role of Corynebacterium non diphtheriae in patients with different pathology].

    PubMed

    Kraeva, L A; Manina, Zh N; Tseneva, G Ia; Radchenko, A G

    2007-01-01

    Bacteriologic examination of 1589 patients showed that, aside from C. diphtheriae, 11% of acute upper respiratory tract infections were caused by other Corynebacterium species. Such bacteria can cause infections of various localizations (bronchitis, pyelonephritis, urethritis, colpitis, dermatitis, arthritis, etc.). C. pseudodiphtheriticum and C. xerosis were isolated from clinical specimens most frequently. Corynebacterium spp. have adhesive, hemolytic, hemagglutinating, and neuraminidase activity; some of them are highly pathogenic. The most virulent, were following species: C. diphtheriae, C. pseudotuberculosis, C. urealyticum, and C. ulcerans. Corynebacterium non diphtheriae were frequently isolated from clinical specimens in association with staphylococci and streptococci. In such cases, factors of pathogenicity and resistance to antibiotics were more pronounced. Strains isolated with association with other bacteria have lost susceptibility to tetracycline, oleandomycin, penicillin, and erythromycin. It is important to be vigilant about bacteria from Corynebacterium genus in clinical settings, and thoroughly study their biologic characteristics, especially in immunocompromised patients.

  16. The Pasteur Hospital as an element of Emile Roux's anti-diphtheria apparatus (1890-1914).

    PubMed

    Opinel, Annick

    2007-01-01

    Before the decisive discovery by Gaston Ramon of a vaccine ("anatoxine diphtérique") in 1923, the fight against diphtheria in France had started in 1894 with the serotherapy approach of Martin and Roux. Emile Roux, director of the Institut Pasteur, developed a dynamic concept of research/production/application expressed in the organization of a specialized hospital, the Hôpital Pasteur, which was constructed near the research laboratories and also incorporated production centres outside Paris in Marnes-la-Coquette. Roux implemented a well-defined project against diphtheria that took account of all of the logistical implications. By associating this therapeutic project with an architectural project, Roux established a coherent anti-diphtheria apparatus.

  17. Exudative pharyngitis possibly due to Corynebacterium pseudodiphtheriticum, a new challenge in the differential diagnosis of diphtheria.

    PubMed Central

    Izurieta, H. S.; Strebel, P. M.; Youngblood, T.; Hollis, D. G.; Popovic, T.

    1997-01-01

    Corynebacterium pseudodiphtheriticum has rarely been reported to cause disease in humans, despite its common presence in the flora of the upper respiratory tract. We report here a case of exudative pharyngitis with pseudomembrane possibly caused by C. pseudodiphtheriticum in a 4-year-old girl. The case initially triggered clinical and laboratory suspicion of diphtheria. Because C. pseudodiphtheriticum can be easily confused with Corynebacterium diphtheriae in Gram stain, clarification of its role in the pathogenesis of exudative pharyngitis in otherwise healthy persons is of public health importance. Simple and rapid screening tests to differentiate C. pseudodiphtheriticum from C. diphtheriae should be performed to prevent unnecessary concern in the community and unnecessary outbreak control measures. PMID:9126447

  18. Causes of low tetanus toxoid vaccination coverage in pregnant women in Lahore district, Pakistan.

    PubMed

    Hasnain, S; Sheikh, N H

    2007-01-01

    To assess the causes of low tetanus toxoid (TT) vaccination coverage in pregnant women a mixture of quantitative and qualitative methods were adopted at the community, primary health care delivery and management levels in Lahore district, Pakistan. Out of a random sample of 362 women who had delivered during the previous 3 months, 87% recalled receiving 2 doses of TT. The main reasons for non-vaccination were poor knowledge about the importance of TT (32% of women) or the place and time to get vaccinated (18%). According to the managers and primary health care medical officers, the main reasons for low coverage were lack of awareness about the importance of vaccination among the public and misconceptions about TT vaccination (e.g. that it was a contraceptive).

  19. Efficacy of Clostridium botulinum types C and D toxoid vaccination in Danish cows.

    PubMed

    Krüger, Monika; Skau, Marie; Shehata, Awad Ali; Schrödl, Wieland

    2013-10-01

    In the present study the efficacy Botulism vaccine (formalinised aluminum hydroxide gel adsorbed toxoid of Clostridium botulinum types C and D) was evaluated in four Danish dairy cows under field conditions. Other four dairy herds were unvaccinated. Blood serum of all animals was analyzed for specific C. botulinum types A, B, C, D and E antibodies using a developed ELISA. Feces of all animals were analyzed for botulinum neurotoxins (BoNTs) and C. botulinum spores. C. botulinum types C and D antibodies were significantly (p < 0.05) increased in vaccinated animals. Vaccination with botulism vaccine significantly reduced (p < 0.001) BoNTs and C. botulinum spores in cattle feces. Our findings represent that C. botulinum vaccination increases specific blood serum antibodies and reduces free BoNTs and C. botulinum spores in feces.

  20. Phase I dose finding studies of an adjuvanted Clostridium difficile toxoid vaccine.

    PubMed

    Greenberg, Richard N; Marbury, Thomas C; Foglia, Ginamarie; Warny, Michel

    2012-03-16

    Fifty healthy adult (18-55 years) and 48 elderly (≥ 65 years) volunteers were randomized to receive a candidate Clostridium difficile toxoid vaccine (2 μg, 10 μg, or 50 μg) or placebo on Days 0, 28, and 56. No volunteer receiving placebo seroconverted. For toxin A, seroconversion by Day 56 (post-dose 2) was observed in 100% of volunteers aged 18-55 years in all dose groups and in 50%, 89%, and 100% of elderly participants in the 2 μg, 10 μg, and 50 μg dose groups, respectively. For both age groups, seroconversion for toxin B was lower than toxin A. There were no safety concerns.

  1. Acceptability of tetanus toxoid vaccine by pregnant women in two health centres in Abidjan (Ivory Coast).

    PubMed

    Ymba, Awa; Perrey, Christophe

    2003-07-28

    A study was conducted in two health centres in Abidjan, Ivory Coast (Abobo and Port Bouet) to compare the knowledge of pregnant women regarding tetanus and hepatitis B and to evaluate the acceptability of tetanus immunisation. A total of 124 women were interviewed. In spite of Information Education Communication (IEC) meetings held by midwives focusing on both diseases, knowledge about tetanus appeared to be substantially higher than that about hepatitis B. The acceptability of tetanus toxoid vaccine was good, the only barrier being the fear of useless injections. However, the risk of tetanus transmission during delivery should be better explained to women, who often give birth at home in the presence of traditional birth assistants. The awareness of men should also be raised by information campaigns, using different local networks.

  2. Tetanus toxoid coverage as an indicator of serological protection against neonatal tetanus.

    PubMed Central

    Deming, Michael S.; Roungou, Jean-Baptiste; Kristiansen, Max; Heron, Iver; Yango, Alphonse; Guenengafo, Alexis; Ndamobissi, Robert

    2002-01-01

    OBJECTIVE: A Multiple-Indicator Cluster Survey (MICS) was conducted at mid-decade in more than 60 developing countries to measure progress towards the year 2000 World Summit for Children goals. These goals included the protection of at least 90% of children against neonatal tetanus through the immunization of their mothers, as measured by tetanus toxoid (TT) coverage. In the Central African Republic (CAR), serological testing was added to the MICS to understand better the relationship between survey estimates of TT coverage and the prevalence of serological protection. METHODS: In the CAR MICS, mothers of children younger than one year of age gave verbal histories of the TT vaccinations they had received, using the MICS TT questionnaire. A subsample of mothers was tested for tetanus antitoxin, using a double-antigen enzyme-linked immunoadsorbent assay (ELISA). Seropositivity was defined as a titre of > or =0.01 IU/ml, and TT coverage was defined as the proportion of mothers protected at delivery, according to their history of TT vaccinations. FINDINGS: Among the 222 mothers in the subsample, weighted TT coverage was 74.4% (95% Confidence Interval (CI); 67.0% - 81.7%) and tetanus antitoxin seroprevalence was 88.7% (95% CI; 83.2% - 94.2%). The weighted median antitoxin titre was 0.35 IU/ml. CONCLUSIONS: Tetanus toxoid coverage in the CAR was lower than the prevalence of serological protection against neonatal tetanus. If this relationship holds for other countries, TT coverage estimates from the MICS may underestimate the extent to which the year 2000 goal for protecting children against neonatal tetanus was reached. We also showed that a high level of serological protection had been achieved in a country facing major public health challenges and resource constraints. PMID:12378286

  3. Interaction of cultured mammalian cells with [125I] diphtheria toxin.

    PubMed Central

    Bonventre, P F; Saelinger, C B; Ivins, B; Woscinski, C; Amorini, M

    1975-01-01

    The characteristics of cell adsorption and pinocytotic uptake of diphtheria toxin by several mammalian cell types were studied. Purified toxin iodinated by a solid-state lactoperoxidase method provided preparations of high specific activity and unaltered biological activity. Dephtheria toxin-sensitive HEp-2 cells and guinea pig macrophage cultures were compared with resistant mouse L-929 cells. At 37 C the resistant cells in monolayer adsorbed and internalized [125I] toxin to a greater extent than did the HEp-2 cell cultures; no significant differences were observed at 5 C. Ammonium chloride protection levels did not alter uptake of toxin by either L-929 OR HEp-2 cells. Biological activity of the iodinated toxin, however, was negated provided the presence of ammonium chloride was maintained. The ammonium salt appears to maintain toxin in a state amenable to antitoxin neutralization. Guinea pig macrophages internalized iodinated toxin to a level 10 times greater than the established cell lines. In spite of the increased uptake of toxin by the endocytic cells, ammonium chloride prevented expression of toxicity. In an artificial system, toxin adsorbed to polystyrene latex spheres and internalized by guinea pig macrophages during phagocytosis did express biological activity. Ammonium chloride afforded some but not total protection against toxin present in the phagocytic vacuoles. The data suggest that two mechanisms of toxin uptake by susceptible cells may be operative. Toxin taken into the cell by a pinocytotic process probably is not ordinarily of physiological significance since it is usually degraded by lysosomal enzymes before it can reach cytoplasmic constituents on which it acts. When large quantities of toxin are pinocytized, toxicity may be expressed before enzymatic degradation is complete. A more specific uptake involving direct passage of the toxin through the plasma membrane may be the mechanism leading to cell death in the majority of instances. PMID

  4. Impaired cellular immune response to tetanus toxoid but not to cytomegalovirus in effectively HAART-treated HIV-infected children.

    PubMed

    Alsina, Laia; Noguera-Julian, Antoni; Fortuny, Clàudia

    2013-05-07

    Despite of highly active antiretroviral therapy, the response to vaccines in HIV-infected children is poor and short-lived, probably due to a defect in cellular immune responses. We compared the cellular immune response (assessed in terms of IFN-γ production) to tetanus toxoid and to cytomegalovirus in a series of 13 HIV-perinatally-infected children and adolescents with optimal immunovirological response to first line antiretroviral therapy, implemented during chronic infection. A stronger cellular response to cytomegalovirus (11 out of 13 patients) was observed, as compared to tetanus toxoid (1 out of 13; p=0.003). These results suggest that the repeated exposition to CMV, as opposed to the past exposition to TT, is able to maintain an effective antigen-specific immune response in stable HIV-infected pediatric patients and strengthen current recommendations on immunization practices in these children.

  5. Seroprevalence of antibody against diphtheria among the population in Khon Kaen province, Thailand.

    PubMed

    Bansiddhi, Hataichanok; Vuthitanachot, Viboonsuk; Vuthitanachot, Chanpim; Prachayangprecha, Slinporn; Theamboonlers, Apiradee; Poovorawan, Yong

    2015-03-01

    To assess diphtheria immunity in the northeastern region of Thailand, a seroepidemiological survey was undertaken in 2011 from 516 healthy individuals (age range 2-87 years) in Khon Kaen province. Diphtheria antitoxin levels were measured by enzyme-linked immunosorbent assay and titers of ≥0.1 IU/mL were considered to be protective antitoxin levels. Among the studied population, 94.8% have fully protective levels. The younger population (age range 2-19 years) has higher diphtheria immunity with seroprotection rates of 96.8% to 97.9%, compared with the adult population. The proportion of protective diphtheria antitoxin levels declines to 88.3% to 91.9% in the middle-aged group (20-50 years), and appeared to be higher again in the older age-group (50-70 years). To avoid epidemic spreading, promoting immunization booster programs will be helpful, especially among the adult population (20-50 years). Finally, this study may serve as a valuable guide in deciding exactly which age-groups should be targeted by such an effort.

  6. Nontoxigenic highly pathogenic clone of Corynebacterium diphtheriae, Poland, 2004-2012.

    PubMed

    Zasada, Aleksandra A

    2013-11-01

    Twenty-five cases of nontoxigenic Corynebacterium diphtheriae infection were recorded in Poland during 2004-2012, of which 18 were invasive. Alcoholism, homelessness, hepatic cirrhosis, and dental caries were predisposing factors for infection. However, for 17% of cases, no concomitant diseases or predisposing factors were found.

  7. Genomic analysis of a nontoxigenic, invasive Corynebacterium diphtheriae strain from Brazil.

    PubMed

    Encinas, Fernando; Marin, Michel A; Ramos, Juliana N; Vieira, Verônica V; Mattos-Guaraldi, Ana Luiza; Vicente, Ana Carolina P

    2015-09-01

    We report the complete genome sequence and analysis of an invasive Corynebacterium diphtheriae strain that caused endocarditis in Rio de Janeiro, Brazil. It was selected for sequencing on the basis of the current relevance of nontoxigenic strains for public health. The genomic information was explored in the context of diversity, plasticity and genetic relatedness with other contemporary strains.

  8. Identification and functional characterization of the NanH extracellular sialidase from Corynebacterium diphtheriae.

    PubMed

    Kim, Seonghun; Oh, Doo-Byoung; Kwon, Ohsuk; Kang, Hyun Ah

    2010-04-01

    Corynebacterium diphtheriae, a pathogenic Gram-positive bacterium, contains sialic acids on its cell surface, but no genes related to sialic acid decoration or metabolism have been reported in C. diphtheriae. In the present study, we have identified a putative sialidase gene, nanH, from C. diphtheriae KCTC3075 and characterized its product for enzyme activity. Interestingly, the recombinant NanH protein was secreted as a catalytically active sialidase into the periplasmic space in Escherichia coli, while the short region at its C-terminus was truncated by proteolysis. We reconstructed a truncated NanH protein (His(6)-NanH(DeltaN)) devoid of its signal sequence as a mature enzyme fused with the 6xHis tag at the N-terminal region. The purified His(6)-NanH(DeltaN) can cleave alpha-2,3- and alpha-2,6-linked sialic acid from sialic acid-containing substrates. In addition, even though the efficiency was low, the recombinant His(6)-NanH(DeltaN) was able to catalyse the transfer of sialic acid using several sialoconjugates as donor, suggesting that the reversible nature of C. diphtheriae NanH can be used for the synthesis of sialyl oligosaccharides via transglycosylation reaction.

  9. Report: seroprevalence of corynebacterium diphtheriae among vaccinated population of Rawalpindi/Islamabad, Pakistan.

    PubMed

    Faryal, Rani; Noreen, Zobia; Tahir, Faheem; Rehman, Zahidur

    2013-05-01

    Diphtheria is a communicable disease of global significance, and its outbreaks have to be reported to the world community under the International Health Regulations (IHR). A pilot seroepidemiological survey was conducted to assess immunity status of diphtheria among healthy individuals of Rawalpindi/Islamabad (Pakistan), who had been administered at least one dose of the vaccine against the disease, as part of childhood vaccination. The study group comprised of 128 healthy subjects, grouped according to the decade representing their age. Antidiphtheria IgG levels were measured by Enzyme Linked Immunosorbent Assay (ELISA) method. The studied sample showed 100% prevalence of diphtheria antitoxin, confirming prior vaccination; however 49.2% exhibited only minimal protection against diphtheria. Full protection was observed in a significantly higher (p=0.013) percentage of males (54.45%) as compared to female subjects (33.33%). Maximum level of serum antibodies were seen in 1-10 year age group (0.195+0.031 IU/mL), which was significantly higher than that recorded in the age group of 11-20 (p=0.024) and above 30 years (p=0.0064). The present results emphasize the need for periodical booster immunization in adolescents and adults, after primary childhood immunization.

  10. Standardization and validation of Vero cell assay for potency estimation of diphtheria antitoxin serum.

    PubMed

    Kumar, Sunil; Kanwar, Sarika; Bansal, Vivek; Sehgal, Rakesh

    2009-10-01

    Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assay and in-vitro Vero cell assay. The assay has also been found to be effective in determining the rising antibody titer in the equines inducted in DATS production. The present study indicated that although biological assays hold the key for final potency estimations till date but in the future scenario in-vitro Vero cell assay may be a good alternative to in-vivo biological assay.

  11. Characterization of OxyR as a negative transcriptional regulator that represses catalase production in Corynebacterium diphtheriae.

    PubMed

    Kim, Ju-Sim; Holmes, Randall K

    2012-01-01

    Corynebacterium diphtheriae and Corynebacterium glutamicum each have one gene (cat) encoding catalase. In-frame Δcat mutants of C. diphtheriae and C. glutamicum were hyper-sensitive to growth inhibition and killing by H(2)O(2). In C. diphtheriae C7(β), both catalase activity and cat transcription decreased ~2-fold during transition from exponential growth to early stationary phase. Prototypic OxyR in Escherichia coli senses oxidative stress and it activates katG transcription and catalase production in response to H(2)O(2). In contrast, exposure of C. diphtheriae C7(β) to H(2)O(2) did not stimulate transcription of cat. OxyR from C. diphtheriae and C. glutamicum have 52% similarity with E. coli OxyR and contain homologs of the two cysteine residues involved in H(2)O(2) sensing by E. coli OxyR. In-frame ΔoxyR deletion mutants of C. diphtheriae C7(β), C. diphtheriae NCTC13129, and C. glutamicum were much more resistant than their parental wild type strains to growth inhibition by H(2)O(2). In the C. diphtheriae C7(β) ΔoxyR mutant, cat transcripts were about 8-fold more abundant and catalase activity was about 20-fold greater than in the C7(β) wild type strain. The oxyR gene from C. diphtheriae or C. glutamicum, but not from E. coli, complemented the defect in ΔoxyR mutants of C. diphtheriae and C. glutamicum and decreased their H(2)O(2) resistance to the level of their parental strains. Gel-mobility shift, DNaseI footprint, and primer extension assays showed that purified OxyR from C. diphtheriae C7(β) bound, in the presence or absence of DTT, to a sequence in the cat promoter region that extends from nucleotide position -55 to -10 with respect to the +1 nucleotide in the cat ORF. These results demonstrate that OxyR from C. diphtheriae or C. glutamicum functions as a transcriptional repressor of the cat gene by a mechanism that is independent of oxidative stress induced by H(2)O(2).

  12. Invasion of endothelial cells and arthritogenic potential of endocarditis-associated Corynebacterium diphtheriae.

    PubMed

    Peixoto, Renata Stavracakis; Pereira, Gabriela Andrade; Sanches dos Santos, Louisy; Rocha-de-Souza, Cláudio Marcos; Gomes, Débora Leandro Rama; Silva Dos Santos, Cintia; Werneck, Lucia Maria Correa; Dias, Alexandre Alves de Souza de Oliveira; Hirata, Raphael; Nagao, Prescilla Emy; Mattos-Guaraldi, Ana Luíza

    2014-03-01

    Although infection by Corynebacterium diphtheriae is a model of extracellular mucosal pathogenesis, different clones have been also associated with invasive infections such as sepsis, endocarditis, septic arthritis and osteomyelitis. The mechanisms that promote C. diphtheriae infection and haematogenic dissemination need further investigation. In this study we evaluated the association and invasion mechanisms with human umbilical vein endothelial cells (HUVECs) and experimental arthritis in mice of endocarditis-associated strains and control non-invasive strains. C. diphtheriae strains were able to adhere to and invade HUVECs at different levels. The endocarditis-associated strains displayed an aggregative adherence pattern and a higher number of internalized viable cells in HUVECs. Transmission electron microscopy (TEM) analysis revealed intracellular bacteria free in the cytoplasm and/or contained in a host-membrane-confined compartment as single micro-organisms. Data showed bacterial internalization dependent on microfilament and microtubule stability and involvement of protein phosphorylation in the HUVEC signalling pathway. A high number of affected joints and high arthritis index in addition to the histopathological features indicated a strain-dependent ability of C. diphtheriae to cause severe polyarthritis. A correlation between the arthritis index and increased systemic levels of IL-6 and TNF-α was observed for endocarditis-associated strains. In conclusion, higher incidence of potential mechanisms by which C. diphtheriae may access the bloodstream through the endothelial barrier and stimulate the production of pro-inflammatory cytokines such as IL-6 and TNF-α, in addition to the ability to affect the joints and induce arthritis through haematogenic spread are thought to be related to the pathogenesis of endocarditis-associated strains.

  13. Higher Tetanus Toxoid Immunity 2 Years After PsA-TT Introduction in Mali

    PubMed Central

    Basta, Nicole E.; Borrow, Ray; Berthe, Abdoulaye; Onwuchekwa, Uma; Dembélé, Awa Traoré Eps; Almond, Rachael; Frankland, Sarah; Patel, Sima; Wood, Daniel; Nascimento, Maria; Manigart, Olivier; Trotter, Caroline L.; Greenwood, Brian; Sow, Samba O.

    2015-01-01

    Background. In 2010, mass vaccination with a then-new meningococcal A polysaccharide–tetanus toxoid protein conjugate vaccine (PsA-TT, or MenAfriVac) was undertaken in 1- to 29-year-olds in Bamako, Mali. Whether vaccination with PsA-TT effectively boosts tetanus immunity in a population with heterogeneous baseline tetanus immunity is not known. We assessed the impact of PsA-TT on tetanus toxoid (TT) immunity by quantifying age- and sex-specific immunity prior to and 2 years after introduction. Methods. Using a household-based, age-stratified design, we randomly selected participants for a prevaccination serological survey in 2010 and a postvaccination survey in 2012. TT immunoglobulin G (IgG) antibodies were quantified and geometric mean concentrations (GMCs) pre- and postvaccination among all age groups targeted for vaccination were compared. The probability of TT IgG levels ≥0.1 IU/mL (indicating short-term protection) and ≥1.0 IU/mL (indicating long-term protection) by age and sex was determined using logistic regression models. Results. Analysis of 793 prevaccination and 800 postvaccination sera indicated that while GMCs were low pre–PsA-TT, significantly higher GMCs in all age–sex strata were observed 2 years after PsA-TT introduction. The percentage with short-term immunity increased from 57.1% to 88.4% (31.3-point increase; 95% confidence interval [CI], 26.6–36.0;, P < .0001) and with long-term immunity increased from 20.0% to 58.5% (38.5-point increase; 95% CI, 33.7–43.3; P < .0001) pre- and postvaccination. Conclusions. Significantly higher TT immunity was observed among vaccine-targeted age groups up to 2 years after Mali's PsA-TT mass vaccination campaign. Our results, combined with evidence from clinical trials, strongly suggest that conjugate vaccines containing TT such as PsA-TT should be considered bivalent vaccines because of their ability to boost tetanus immunity. PMID:26553691

  14. Chitosan-HPMC-blended microspheres as a vaccine carrier for the delivery of tetanus toxoid.

    PubMed

    Arthanari, Saravanakumar; Mani, Ganesh; Peng, Mei Mei; Jang, Hyun Tae

    2016-01-01

    The purpose of this research was to develop a suitable and alternate adjuvant for the tetanus toxoid (TT) vaccine that induces long term immunity after a single-dose immunization. In our study, the preformulation studies were carried out by using different ratios (7/3, 8/2, and 9/1) of chitosan-hydroxypropyl methylcellulose (HPMC)-blended empty microspheres. Moreover, TT was stabilized with heparin (at heparin concentrations of 1%, 2%, 3%, and 4% w/v) and encapsulated in ideal chitosan - HPMC (CHBMS) microspheres, by the water-in-oil-in-water (W/O/W) multiple emulsion method. The vaccine entrapment and the in vitro release efficiency of the CHBMS was evaluated for a period of 90 days. The release of antigens from the microspheres was determined by ELISA. Antigen integrity was investigated by SDS-PAGE. From the optimization studies, it was found that a chitosan/HPMC ratio of 8/2 produced a good yield, with microspheres that were spherical, regular and uniformly-sized. In the CHBMS, a heparin concentration of 3% w/v resulted in well-sustained antigen delivery for a period of 90 days. It was found that the characteristics of initial release could be observed in 2 days, followed by a constant release, and an almost 100% complete release in 90 days. From the in vitro release characteristics, the ideal batch of CHBMS (3% w/v heparin) was evaluated for in vivo studies by the antibody induction method. The antibody levels were measured for different combinations for the period of 9 months, and finally, with a second booster dose after 1 year. In conclusion, it was observed that CHBMS (combination-1) resulted in the antibody level of 4.5 IU/mL of guinea pig serum, and the level was 3.5 IU/mL for the Central Research Institute's alum-adsorbed tetanus toxoid (CRITT) (combination 2), after 1 year, with a second booster dose. This novel approach of using CHBMS may have potential advantages for single-step immunization with vaccines.

  15. Comparison of four molecular typing methods for characterization of Corynebacterium diphtheriae and determination of transcontinental spread of C. diphtheriae based on BstEII rRNA gene profiles.

    PubMed

    De Zoysa, Aruni; Hawkey, Peter; Charlett, Andre; Efstratiou, Androulla

    2008-11-01

    The diphtheria epidemic in the Russian Federation in the 1990s made diphtheria a focus of global concern once again. The development of rapid and reproducible typing methods for the molecular characterization of Corynebacterium diphtheriae has become a priority in order to be able to monitor the spread of this important pathogen on a global scale. We report on a comparison of four molecular typing methods (ribotyping, pulsed-field gel electrophoresis [PFGE], random amplification of polymorphic DNA [RAPD], and amplified fragment length polymorphism [AFLP]) for the characterization of C. diphtheriae strains. Initially, 755 isolates originating from 26 countries were analyzed by ribotyping. One strain of each ribotype was then randomly chosen and characterized by PFGE, RAPD, and AFLP. In order to ascertain whether the Eastern European epidemic ribotype could be further discriminated, 10 strains of ribotype D1 (the epidemic ribotype) from different geographical regions were randomly chosen and subjected to analysis by PFGE, RAPD, and AFLP. The results revealed that ribotyping is highly discriminatory and reproducible and is currently the method of choice for typing C. diphtheriae. PFGE and AFLP were less discriminatory than ribotyping and RAPD. An assessment of the transcontinental spread of the organism showed that several genotypes of C. diphtheriae circulated on different continents of the world and that each outbreak was caused by a distinct clone. The ribotypes seen in Europe appeared to be distinct from those seen elsewhere, and certain ribotypes appeared to be unique to particular countries.

  16. A novel approach to generate a recombinant toxoid vaccine against Clostridium difficile.

    PubMed

    Donald, Robert G K; Flint, Mike; Kalyan, Narender; Johnson, Erik; Witko, Susan E; Kotash, Cheryl; Zhao, Ping; Megati, Shakuntala; Yurgelonis, Irina; Lee, Phillip Kwok; Matsuka, Yury V; Severina, Elena; Deatly, Anne; Sidhu, Mini; Jansen, Kathrin U; Minton, Nigel P; Anderson, Annaliesa S

    2013-07-01

    The Clostridium difficile toxins A and B are primarily responsible for symptoms of C. difficile associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of C. difficile that lacks the toxin genes tcdA and tcdB. TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from C. difficile transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10 000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a C. difficile toxoid vaccine.

  17. [Cloning and expression of a hemolysin gene of Aeromonas hydrophila and the immunogenicity of the toxoid].

    PubMed

    Zhang, Cuijuan; Yu, Zhouliang; Tian, Liying; Zhao, Baohua

    2009-02-01

    According to the GenBank sequences (GenBank Accession No. AF539467), one pair of primers was designed to amplify hly gene of Aeromonas hydrophila by PCR. After sequencing, homology analysis indicated that a DNA fragment of 1485 bp was amplified from isolated DNA from Aeromonas hydrophila, and it shared more than 99% homology in nucleotide sequence compared with other reference strains in GenBank. The gene was cloned in pET-28a vector to construct a recombinant plasmid pET-28a-hly, which was transformed into Escherichia coli BL21 (DE3), and the recombinant strain BL21(DE3)(pET-28a-hly) was obtained. The hemolysin was highly expressed when the recombinant strain BL21 (DE3) (pET-28a-hly) was induced by IPTG. The expressed protein was 56 kD as estimated by 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The immunogenicity of the expressed Hly protein was confirmed by Western blotting. Mice were immunized with inactivated whole bacteria vaccine and the genetic engineering vaccines showing promise that all these vaccines have a high protective ability. The results showed that the recombinant strain BL21 (DE3)(pET-28a-hly) could be candidate of hemolysin toxoid vaccine to provide protective immunity against diseases caused by Aeromonas hydrophila.

  18. Optimization of tetanus toxoid ammonium sulfate precipitation process using response surface methodology.

    PubMed

    Brgles, Marija; Prebeg, Pero; Kurtović, Tihana; Ranić, Jelena; Marchetti-Deschmann, Martina; Allmaier, Günter; Halassy, Beata

    2016-10-02

    Tetanus toxoid (TTd) is a highly immunogenic, detoxified form of tetanus toxin, a causative agent of tetanus disease, produced by Clostridium tetani. Since tetanus disease cannot be eradicated but is easily prevented by vaccination, the need for the tetanus vaccine is permanent. The aim of this work was to investigate the possibility of optimizing TTd purification, i.e., ammonium sulfate precipitation process. The influence of the percentage of ammonium sulfate, starting amount of TTd, buffer type, pH, temperature, and starting purity of TTd on the purification process were investigated using optimal design for response surface models. Responses measured for evaluation of the ammonium sulfate precipitation process were TTd amount (Lf/mL) and total protein content. These two parameters were used to calculate purity (Lf/mgPN) and the yield of the process. Results indicate that citrate buffer, lower temperature, and lower starting amount of TTd result in higher purities of precipitates. Gel electrophoresis combined with matrix-assisted laser desorption ionization-mass spectrometric analysis of precipitates revealed that there are no inter-protein cross-links and that all contaminating proteins have pIs similar to TTd, so this is most probably the reason for the limited success of purification by precipitation.

  19. Usefulness of 16S rDNA sequencing for the diagnosis of infective endocarditis caused by Corynebacterium diphtheriae.

    PubMed

    Pathipati, Padmaja; Menon, Thangam; Kumar, Naveen; Francis, Thara; Sekar, Prem; Cherian, Kotturathu Mammen

    2012-08-01

    We report a rare case of infective endocarditis caused by Corynebacterium diphtheriae in an 8-year-old boy, 2 years after a right ventricular outflow tract reconstruction with a bovine Contegra valved conduit. The patient recovered well after an RV-PA conduit enblock explantation and replacement with an aortic homograft with antibiotic treatment. All bacteriological cultures of excised tissue and blood were negative. The aetiological agent was identified as C. diphtheriae subsp. gravis by 16s rDNA sequencing.

  20. A PCR for dtxR gene: application to diagnosis of non-toxigenic and toxigenic Corynebacterium diphtheriae.

    PubMed

    Pimenta, Fabricia P; Matias, Gisele A M; Pereira, Gabriela A; Camello, Thereza C F; Alves, Gabriela B; Rosa, Ana C P; Hirata, Raphael; Mattos-Guaraldi, Ana L

    2008-06-01

    The significant rise in the percentage of adults susceptible to diphtheria and the emergence of non-toxigenic Corynebacterium diphtheriae strains as the causative agent of endocarditis and other systemic infections emphasize the need for alternative laboratory diagnostic procedures. In this study, for the first time, the value of a species-specific PCR assay that targets the dtxR gene is documented as a procedure for differentiating C. diphtheriae from Corynebacterium-like colonies. The results of the PCR-dtxR were all positive for 91 C. diphtheriae (54 non-toxigenic and 37 toxigenic) strains. PCR-dtxR completely correlated with the standard biochemical and commercial identification for all C. diphtheriae strains tested. Conversely, the PCR-dtxR results were negative in 100% of the 111 non-diphtherial Gram-positive rod strains obtained during identification procedures in a hospital laboratory. Thus, the PCR-dtxR assay emerged as viable, cost-effective screening method for C. diphtheriae laboratory identification.

  1. Lack of significant differences in immunity against diphtheria between populations of Eastern and Western regions of Poland.

    PubMed

    Chudnicka, Alina; Walory, Jarosław

    2003-01-01

    Incidents of diphtheria in countries which were formerly part of the Soviet Union (Ukraine, Russia and Belorus) resulted in the need to evaluate thoroughly the effectiveness of preventive vaccination in Poland, especially in the border regions of the country where the biggest migration of population can be observed. The aim of this work was a comparison of the immunity to diphtheria in two geographically different regions of Poland--eastern (Lublin) and western (Zielona Gora) ones. It showed immunoprophylaxis to diphtheria that was implemented on these areas. Diphtheria antitoxin level (IgG) was determined with application of the ELISA method in 1236 (529/707) people. No significant differences were found in the level of antibodies in the groups < 2 years of age and > 19 years of age in people below the protective titre (0.1 IU/ml). The difference occurring in the interval between 2nd and 18th year of life (in western Poland 7.6% and in eastern Poland 16%) may result from different implementation of the vaccination program in these regions (booster doses). Recommendations for vaccination to diphtheria in people over 25 years of age should be implemented especially in the frontier regions of Poland adjoining countries threatened with diphtheria occurrence.

  2. Analysis of novel iron-regulated, surface-anchored hemin-binding proteins in Corynebacterium diphtheriae.

    PubMed

    Allen, Courtni E; Burgos, Jonathan M; Schmitt, Michael P

    2013-06-01

    Corynebacterium diphtheriae utilizes hemin and hemoglobin (Hb) as iron sources during growth in iron-depleted environments, and recent studies have shown that the surface-exposed HtaA protein binds both hemin and Hb and also contributes to the utilization of hemin iron. Conserved (CR) domains within HtaA and in the associated hemin-binding protein, HtaB, are required for the ability to bind hemin and Hb. In this study, we identified and characterized two novel genetic loci in C. diphtheriae that encode factors that bind hemin and Hb. Both genetic systems contain two-gene operons that are transcriptionally regulated by DtxR and iron. The gene products of these operons are ChtA-ChtB and ChtC-CirA (previously DIP0522-DIP0523). The chtA and chtB genes are carried on a putative composite transposon associated with C. diphtheriae isolates that dominated the diphtheria outbreak in the former Soviet Union in the 1990s. ChtA and ChtC each contain a single N-terminal CR domain and exhibit significant sequence similarity to each other but only limited similarity with HtaA. The chtB and htaB gene products exhibited a high level of sequence similarity throughout their sequences, and both proteins contain a single CR domain. Whole-cell binding studies as well as protease analysis indicated that all four of the proteins encoded by these two operons are surface exposed, which is consistent with the presence of a transmembrane segment in their C-terminal regions. ChtA, ChtB, and ChtC are able to bind hemin and Hb, with ChtA showing the highest affinity. Site-directed mutagenesis showed that specific tyrosine residues within the ChtA CR domain were critical for hemin and Hb binding. Hemin iron utilization assays using various C. diphtheriae mutants indicate that deletion of the chtA-chtB region and the chtC gene has no affect on the ability of C. diphtheriae to use hemin or Hb as iron sources; however, a chtB htaB double mutant exhibits a significant decrease in hemin iron use

  3. Expression and purification of truncated diphtheria toxin, DT386, in Escherichia coli: An attempt for production of a new vaccine against diphtheria

    PubMed Central

    Shafiee, Fatemeh; Rabbani, Mohammad; Behdani, Mahdi; Jahanian-Najafabadi, Ali

    2016-01-01

    The aim of this study was to produce a recombinant protein consisting of the catalytic and translocation domains of diphtheria toxin for its later application as a vaccine candidate against Corynebacterium diphtheria. To achieve this goal, at first, the amino acid sequence of DT386 was used for prediction of T and B cell epitopes using on-line servers. The DT386 coding sequence was synthesized and subcloned into the NcoI and XhoI sites of pET28a plasmid and recombinant pET28a plasmid was used to transform Escherichia coli BL21 (DE3) host cells. Afterwards, recombinant cells were selected and subjected to induction of expression by 1 mM isopropyl β-D-1-thiogalactopyranoside, (IPTG). Expression of the desired protein was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, and finally, the recombinant protein was purified using nickel affinity chromatography. The results of epitope prediction using on-line servers established the ability of DT386 for stimulation of immune system against diphtheria toxin. Restriction digestion of the recombinant plasmids using NcoI and XhoI enzymes confirmed the fidelity of cloning by producing a band of about 1200 bp. SDS-PAGE analysis following induction of expression and also purification step confirmed the expression of the desired protein by showing a band of about 45 kDa. In addition, Western blot analysis using anti-6X-His antibody confirmed the identity of the expected protein. In conclusion, in the present study we amplified and cloned the coding sequence of DT386 fragment, followed by its expression by E. coli BL21 (DE3) cells. Then, the expressed protein was purified and will be used for later studies of evaluation of its immunogenic properties. PMID:27920826

  4. Age-specific seroprevalence of poliomyelitis, diphtheria and tetanus antibodies in Spain.

    PubMed Central

    Pachón, I.; Amela, C.; De Ory, F.

    2002-01-01

    In 1996, a seroepidemiological study was undertaken in Spain, with the main aim of estimating the population's immunity against poliomyelitis, tetanus and diphtheria. A population-based cross-sectional study was conducted, covering the population aged 2-39 years. The sample was stratified by age and rural-urban environment, and informed consent obtained to take blood specimens from subjects attending phlebotomy centres. The study included 3,932 persons and the prevalence of antibodies against all three types of poliovirus exceeded 94% across all age groups. From a high of 96% in subjects under the age of 15 years, immunity against diphtheria steadily declined to a low of 32.3% in subjects aged 30-39 years. Similarly, tetanus antitoxin concentrations indicating basic protection were present in 98.9% of the under-14 years age group; thereafter, immunity declined, until reaching 54.6% in the 30-39 years age group. PMID:12558336

  5. Antibody responses to natural rattlesnake envenomation and a rattlesnake toxoid vaccine in horses.

    PubMed

    Gilliam, Lyndi L; Carmichael, Robert C; Holbrook, Todd C; Taylor, Jennifer M; Ownby, Charlotte L; McFarlane, Dianne; Payton, Mark E

    2013-05-01

    Antivenom antibody titers following administration of rattlesnake venom for antivenom production in horses are well documented; however, antivenom antibody titers following natural rattlesnake envenomation in horses are not. Antibody titers produced in response to the commercially available rattlesnake venom vaccine are also not published. Our study objectives were to measure antivenom antibody titers in rattlesnake-bitten horses and compare them to titers in horses vaccinated with the rattlesnake venom vaccine. Additionally, titers were compared in pregnant versus nonpregnant horses to assess the affect of pregnancy on vaccine response and were measured pre- and postsuckle in foals of vaccinated mares to detect passive transfer of vaccine immunoglobulins. Blood samples were collected from 16 rattlesnake-bitten horses. Thirty-six horses (11 pregnant mares, 12 nonpregnant mares, 13 geldings) were vaccinated using a Crotalus atrox venom toxoid vaccine. Blood was collected before administering each vaccination and 30 days following the third vaccination. Blood was collected from foals of vaccinated mares pre- and postsuckle. All serum was assayed for anti-Crotalus atrox venom antibodies using an enzyme-linked immunosorbent assay (ELISA). Rattlesnake-bitten horses had higher (P = 0.001) titers than vaccinated horses. There was no significant difference between titers in vaccinated pregnant versus nonpregnant horses. One mare had a positive titer at foaling, and the foals had positive postsuckle titers. Antivenom antibody titer development was variable following natural envenomation and vaccination, and vaccine-induced titers were lower than natural envenomation titers. Further studies are required to determine if natural or vaccine antivenom antibody titers reduce the effects of envenomation.

  6. CD4 T-helper cell cytokine phenotypes and antibody response following tetanus toxoid booster immunization.

    PubMed

    Livingston, Kimberly A; Jiang, Xiaowen; Stephensen, Charles B

    2013-04-30

    Routine methods for enumerating antigen-specific T-helper cells may not identify low-frequency phenotypes such as Th2 cells. We compared methods of evaluating such responses to identify tetanus toxoid- (TT) specific Th1, Th2, Th17 and IL10(+) cells. Eight healthy subjects were given a TT booster vaccination. Blood was drawn before, 3, 7, 14, and 28days after vaccination and peripheral blood mononuclear cells (PBMC) were cultured for 7days with TT, negative control (diluent), and a positive control (Staphylococcus enterotoxin B [SEB]). Activation markers (CD25 and CD69) were measured after 44h (n=8), cytokines in supernatant after 3 and 7days, and intracellular cytokine staining (ICS) of proliferated cells (identified by dye dilution) after 7days (n=6). Vaccination increased TT-specific expression of CD25 and CD69 on CD3(+)CD4(+) lymphocytes, and TT-specific proliferation at 7, 14 and 28days post vaccination. Vaccination induced TT-specific Th1 (IFN-γ, TNF-α, and IL-2) Th2 (IL-13, IL-5, and IL-4), Th17 (IL-17A) and IL-10(+) cells as measured by ICS. TT-specific Th1 cells were the most abundant (12-15% of all TT-specific CD4(+) T-cells) while IL10(+) (1.8%) Th17 (1.1%) and Th2 cells (0.2-0.6%) were less abundant. TT-specific cytokine concentrations in PBMC supernatants followed the same pattern where a TT-specific IL-9 response was also seen. In conclusion, TT booster vaccination induced a broad T-helper cell response. This method of evaluating cytokine phenotypes may be useful in examining the impact of nutrition and environmental conditions on the plasticity of T-helper cell memory responses.

  7. Tetanus toxoid vaccine: elimination of neonatal tetanus in selected states of India.

    PubMed

    Verma, Ramesh; Khanna, Pardeep

    2012-10-01

    Tetanus is caused by a neurotoxin produced by Clostridium tetani (C. tetani), a spore-forming bacterium. Infection begins when tetanus spores are introduced into damaged tissue. Tetanus is characterized by muscle rigidity and painful muscle spasms caused by tetanus toxin's blockade of inhibitory neurons that normally oppose and modulate the action of excitatory motor neurons. Maternal and neonatal tetanus (MNT) are caused by unhygienic methods of delivery, abortion, or umbilical-cord care. Maternal and neonatal tetanus are both forms of generalized tetanus and have similar clinical courses. About 90% of neonates with tetanus develop symptoms in the first 3-14 d of life, mostly on days 6-8, distinguishing neonatal tetanus from other causes of neonatal mortality which typically occur during the first two days of life. Overall case fatality rates for patients admitted to the hospital with neonatal tetanus in developing countries are 8-50%, while the fatality rate can be as high as 100% without hospital care. Tetanus toxoid (TT) vaccination of pregnant women to prevent neonatal tetanus was included in WHO's Expanded Program on Immunization (EPI) a few years after its inception in 1974. In 2000, WHO, UNICEF, and UNFPA formed a partnership to relaunch efforts toward this goal, adding the elimination of maternal tetanus as a program objective, and setting a new target date of 2005. By February 2007, 40 countries had implemented tetanus vaccination campaigns in high-risk areas, targeting more than 94 million women, and protecting more than 70 million subjects with at least two doses of TT. In 2011, 653 NT cases were reported in India compared with 9313 in 1990. As of February 2012, 25 countries and 15 States and Union Territories of India, all of Ethiopia except Somaliland, and almost 29 of 34 provinces in Indonesia have been validated to have eliminated MNT.

  8. Meningococcal vaccine A,C,W135,Y: conjugated to tetanus toxoid.

    PubMed

    2013-12-01

    A meningococcal vaccine conjugated to protein CRM 197 (Menveo) is the standard vaccine for immunisation against invasive meningococcal infections caused by serogroups A, C, W135 andY, beginning at age 2 years. Nimenrix, another vaccine against meningococcal groups A, C,W135 and Y, conjugated to tetanus toxoid, was authorised for use in the European Union, starting at age 1 year. The two tetravalent meningococcal conjugate vaccines have not been compared in head-to-head trials. Four immunogenicity studies compared the tetravalent conjugate vaccine Nimenrix with an unconjugated tetravalent meningococcal vaccine in children and adults aged 2 to 55 years. The results showed that Nimenrix was more immunogenic than the unconjugated vaccine. Two immunogenicity studies showed that Nimenrix was at least as immunogenic as monovalent (group C) meningococcal conjugate vaccines in children aged from 1 to 2 years and from 2 to 10 years. In one study, prior vaccination with an unconjugated tetravalent meningococcal vaccine had little impact on the immunogenicity of a booster dose of the conjugate vaccine Nimenrix. Concomitant administration with other vaccines does not affect the immunogenicity of Nimenrix. Nimenrix causes more frequent local and systemic adverse reactions than the unconjugated tetravalent meningococcal vaccine and monovalent group C meningococcal conjugate vaccines. In children over 2 years of age, Nimenrix has no advantages over Menveo for vaccination against meningococcal serogroups A, C, W135 andY. In contrast, between the ages of 1 and 2 years, Nimenrix is the only vaccine with established immunogenicity. In addition, it has an acceptable harm-benefit balance.

  9. Seroprevalence of tetanus toxoid antibody and booster vaccination efficacy in Japanese travelers.

    PubMed

    Mizuno, Yasutaka; Yamamoto, Akihiko; Komiya, Takako; Takeshita, Nozomi; Takahashi, Motohide

    2014-01-01

    Tetanus can be prevented by vaccination, which is especially important for overseas travelers. However, despite booster vaccination every 10 years being recommended, most Japanese adults do not receive it in the absence of physical injury or overseas travel. We aimed to investigate the level of protective immunity against tetanus among Japanese travelers, which may provide valuable information for formulating booster vaccination recommendations. 113 Japanese travelers given tetanus toxoid were recruited. The collected samples included paired samples prior to and 3-5 weeks after receiving the booster vaccination. Travelers who did not return and those lacking sample collection at the second visit were excluded. Finally, 96 paired blood samples were collected. History of immunization against tetanus, including DPT and DT vaccines, was determined from interviews or immunization records. The pre-vaccination geometric mean titer for the 96 participants was 1.07 IU/mL; 76% had a protective antitoxin level (>0.1 IU/mL), and 50% had a long-term protective antitoxin level (>1.0 IU/mL). Most participants <40 years old had protective immunity without receiving booster vaccination, whereas only 30.8% of those >50 years of age had protective immunity. Among the 23 participants without protective antitoxin levels (<0.1 IU/mL), booster vaccination was efficient in 100% of those <40 years but in only 28.6% of those >50 years of age. Although the tetanus antitoxin level decreases with age, booster vaccination helped to achieve an adequate protective antitoxin levels in Japanese travelers <40 years of age. Furthermore, the individuals who have never been vaccinated against tetanus especially in those >50 years old need to obtain protective immunity against tetanus according to a basic immunization schedule to prevent tetanus in travelers and residents of Japan.

  10. Autoradiographic assay of mutants resistant to diphtheria toxin in mammalian cells in vitro

    SciTech Connect

    Ronen, A.; Gingerich, J.D.; Duncan, A.M.V.; Heddle, J.A.

    1984-10-01

    Diptheria toxin kills mammalian cells by ribosylating elongation factor 2, a protein factor necessary for protein synthesis. The frequency of cells able to form colonies in the presence of the toxin can be used as an assay for mutation to diphtheria toxin resistance. Resistance to diphtheria toxin can also be detected autoradiographically in cells exposed to (/sup 3/H)leucine after treatment with the toxin. In cultures of Chinese hamster ovary cells, the frequency of such resistant cells is increased by exposure of the cells to ..gamma..-rays, ultraviolet light, ethylnitrosourea, mitomycin c, ethidium bromide, and 5-bromo-2'-deoxyuridine in a dose- and time-dependent manner. The resistant cells form discrete microcolonies if they are allowed to divide several times before intoxication which indicates that they are genuine mutants. The assay is potentially adaptable to any cell population that can be intoxicated with diphtheria toxin and labeled with (/sup 3/H)leucine, whether or not the cells can form colonies. It may be useful, therefore, for measuring mutation rates in slowly growing or nondividing cell populations such as breast, brain, and liver, as well as in cells that do divide but cannot be readily cloned, such as the colonic epithelium. 23 references, 6 figures.

  11. [The use of MLVA for Corynebacterium diphtheriae genotyping--preliminary studies].

    PubMed

    Zasada, Aleksandra A; Jagielski, Marek; Rzeczkowska, Magdalena; Januszkiewicz, Aleksandra

    2011-01-01

    The complete genome sequence of strain NCTC 13129 C. diphtheriae were investigated in order to identify tandem repeats (VNTR). From 75 VNTR loci identified in the genome 14 were selected. Primers were designed and PCR conditions were optimized for amplification of the selected VNTR markers. Preliminary studies of usefulness of selected VNTR markers were conducted using a group of 28 C. diphtheriae strains. From 14 markers 8 were regarded as potentially useful. The diversity of individual markers ranged from 1 to 6 alleles (Simpson index from 0 to 0,746). No diversity were observed for 3 VNTR markers but it could be a results of too small group of strains analyzed in the tests. Simpson diversity index calculated for all the markers tested on 28 strains was 0,87. Results of the preliminary studies showed usefulness of MLVA for C. diphtheriae genotyping. Nevertheless, confirmation of reliability of the method should be done using a large group of strains. Moreover, the method should be compared with other genotyping methods.

  12. Semicarbazone EGA Inhibits Uptake of Diphtheria Toxin into Human Cells and Protects Cells from Intoxication

    PubMed Central

    Schnell, Leonie; Mittler, Ann-Katrin; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

    2016-01-01

    Diphtheria toxin is a single-chain protein toxin that invades human cells by receptor-mediated endocytosis. In acidic endosomes, its translocation domain inserts into endosomal membranes and facilitates the transport of the catalytic domain (DTA) from endosomal lumen into the host cell cytosol. Here, DTA ADP-ribosylates elongation factor 2 inhibits protein synthesis and leads to cell death. The compound 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA) has been previously shown to protect cells from various bacterial protein toxins which deliver their enzymatic subunits from acidic endosomes to the cytosol, including Bacillus anthracis lethal toxin and the binary clostridial actin ADP-ribosylating toxins C2, iota and Clostridium difficile binary toxin (CDT). Here, we demonstrate that EGA also protects human cells from diphtheria toxin by inhibiting the pH-dependent translocation of DTA across cell membranes. The results suggest that EGA might serve for treatment and/or prevention of the severe disease diphtheria. PMID:27428999

  13. Regulation and activity of a zinc uptake regulator, Zur, in Corynebacterium diphtheriae.

    PubMed

    Smith, Kelsy F; Bibb, Lori A; Schmitt, Michael P; Oram, Diana M

    2009-03-01

    Regulation of metal ion homeostasis is essential to bacterial cell survival, and in most species it is controlled by metal-dependent transcriptional regulators. In this study, we describe a Corynebacterium diphtheriae ferric uptake regulator-family protein, Zur, that controls expression of genes involved in zinc uptake. By measuring promoter activities and mRNA levels, we demonstrate that Zur represses transcription of three genes (zrg, cmrA, and troA) in zinc-replete conditions. All three of these genes have similarity to genes involved in zinc uptake. Transcription of zrg and cmrA was also shown to be regulated in response to iron and manganese, respectively, by mechanisms that are independent of Zur. We demonstrate that the activity of the zur promoter is slightly decreased under low zinc conditions in a process that is dependent on Zur itself. This regulation of zur transcription is distinctive and has not yet been described for any other zur. An adjacent gene, predicted to encode a metal-dependent transcriptional regulator in the ArsR/SmtB family, is transcribed from a separate promoter whose activity is unaffected by Zur. A C. diphtheriae zur mutant was more sensitive to peroxide stress, which suggests that zur has a role in protecting the bacterium from oxidative damage. Our studies provide the first evidence of a zinc specific transcriptional regulator in C. diphtheriae and give new insights into the intricate regulatory network responsible for regulating metal ion concentrations in this toxigenic human pathogen.

  14. Pathogenic properties of a Corynebacterium diphtheriae strain isolated from a case of osteomyelitis.

    PubMed

    Peixoto, Renata Stavracakis; Hacker, Elena; Antunes, Camila Azevedo; Weerasekera, Dulanthi; Dias, Alexandre Alves de Souza de Oliveira; Martins, Carlos Alberto; Hirata, Raphael; Santos, Kátia Regina Netto Dos; Burkovski, Andreas; Mattos-Guaraldi, Ana Luíza

    2016-11-01

    Corynebacterium diphtheriae is typically recognized as a colonizer of the upper respiratory tract (respiratory diphtheria) and the skin (cutaneous diphtheria). However, different strains of Corynebacteriumdiphtheriae can also cause invasive infections. In this study, the characterization of a non-toxigenic Corynebacteriumdiphtheriae strain (designated BR-INCA5015) isolated from osteomyelitis in the frontal bone of a patient with adenoid cystic carcinoma was performed. Pathogenic properties of the strain BR-INCA5015 were tested in a Caenorhabditis elegans survival assay showing strong colonization and killing by this strain. Survival rates of 3.8±2.7 %, 33.6±7.3 % and 0 % were observed for strains ATCC 27010T, ATCC 27012 and BR-INCA5015, respectively, at day 7. BR-INCA5015 was able to colonize epithelial cells, showing elevated capacity to adhere to and survive within HeLa cells compared to other Corynebacteriumdiphtheriae isolates. Intracellular survival in macrophages (THP-1 and RAW 264.7) was significantly higher compared to control strains ATCC 27010T (non-toxigenic) and ATCC 27012 (toxigenic). Furthermore, the ability of BR-INCA5015 to induce osteomyelitis was confirmed by in vivo assay using Swiss Webster mice.

  15. Characterization of C-S Lyase from C. diphtheriae: A Possible Target for New Antimicrobial Drugs

    PubMed Central

    2013-01-01

    The emergence of antibiotic resistance in microbial pathogens requires the identification of new antibacterial drugs. The biosynthesis of methionine is an attractive target because of its central importance in cellular metabolism. Moreover, most of the steps in methionine biosynthesis pathway are absent in mammals, lowering the probability of unwanted side effects. Herein, detailed biochemical characterization of one enzyme required for methionine biosynthesis, a pyridoxal-5′-phosphate (PLP-) dependent C-S lyase from Corynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria, has been performed. We overexpressed the protein in E. coli and analyzed substrate specificity, pH dependence of steady state kinetic parameters, and ligand-induced spectral transitions of the protein. Structural comparison of the enzyme with cystalysin from Treponema denticola indicates a similarity in overall folding. We used site-directed mutagenesis to highlight the importance of active site residues Tyr55, Tyr114, and Arg351, analyzing the effects of amino acid replacement on catalytic properties of enzyme. Better understanding of the active site of C. diphtheriae C-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel inhibitors as antibacterial therapeutics. PMID:24106714

  16. Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

    PubMed

    Katsukawa, Chihiro; Komiya, Takako; Umeda, Kaoru; Goto, Minami; Yanai, Tokuma; Takahashi, Motohide; Yamamoto, Akihiko; Iwaki, Masaaki

    2016-03-01

    Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans.

  17. A glycoconjugate of Haemophilus influenzae Type b capsular polysaccharide with tetanus toxoid protein: hydrodynamic properties mainly influenced by the carbohydrate

    PubMed Central

    Abdelhameed, Ali Saber; Adams, Gary G.; Morris, Gordon A.; Almutairi, Fahad M.; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E.

    2016-01-01

    Three important physical properties which may affect the performance of glycoconjugate vaccines against serious disease are molar mass (molecular weight), heterogeneity (polydispersity), and conformational flexibility in solution. The dilute solution behaviour of native and activated capsular polyribosylribitol (PRP) polysaccharides extracted from Haemophilus influenzae type b (Hib), and the corresponding glycoconjugate made by conjugating this with the tetanus toxoid (TT) protein have been characterized and compared using a combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge with viscometry. The weight average molar mass of the activated material was considerably reduced (Mw ~ 0.24 × 106 g.mol−1) compared to the native (Mw ~ 1.2 × 106 g.mol−1). Conjugation with the TT protein yielded large polydisperse structures (of Mw ~ 7.4 × 106 g.mol−1), but which retained the high degree of flexibility of the native and activated polysaccharide, with frictional ratio, intrinsic viscosity, sedimentation conformation zoning behaviour and persistence length all commensurate with highly flexible coil behaviour and unlike the previously characterised tetanus toxoid protein (slightly extended and hydrodynamically compact structure with an aspect ratio of ~3). This non-protein like behaviour clearly indicates that it is the carbohydrate component which mainly influences the physical behaviour of the glycoconjugate in solution. PMID:26915577

  18. Solution conformation and flexibility of capsular polysaccharides from Neisseria meningitidis and glycoconjugates with the tetanus toxoid protein

    NASA Astrophysics Data System (ADS)

    Abdelhameed, Ali Saber; Morris, Gordon A.; Almutairi, Fahad; Adams, Gary G.; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E.

    2016-10-01

    The structural integrity of meningococcal native, micro-fluidized and activated capsular polysaccharides and their glycoconjugates – in the form most relevant to their potential use as vaccines (dilute solution) - have been investigated with respect to their homogeneity, conformation and flexibility. Sedimentation velocity analysis showed that the polysaccharide size distributions were generally bimodal with some evidence for higher molar mass forms at higher concentration. Weight average molar masses Mw where lower for activated polysaccharides. Conjugation with tetanus toxoid protein however greatly increased the molar mass and polydispersity of the final conjugates. Glycoconjugates had an approximately unimodal log-normal but broad and large molar mass profiles, confirmed by sedimentation equilibrium “SEDFIT MSTAR” analysis. Conformation analysis using HYDFIT (which globally combines sedimentation and viscosity data), “Conformation Zoning” and Wales-van Holde approaches showed a high degree of flexibility – at least as great as the unconjugated polysaccharides, and very different from the tetanus toxoid (TT) protein used for the conjugation. As with the recently published finding for Hib-TT complexes, it is the carbohydrate component that dictates the solution behaviour of these glycoconjugates, although the lower intrinsic viscosities suggest some degree of compaction of the carbohydrate chains around the protein.

  19. A glycoconjugate of Haemophilus influenzae Type b capsular polysaccharide with tetanus toxoid protein: hydrodynamic properties mainly influenced by the carbohydrate.

    PubMed

    Abdelhameed, Ali Saber; Adams, Gary G; Morris, Gordon A; Almutairi, Fahad M; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E

    2016-02-26

    Three important physical properties which may affect the performance of glycoconjugate vaccines against serious disease are molar mass (molecular weight), heterogeneity (polydispersity), and conformational flexibility in solution. The dilute solution behaviour of native and activated capsular polyribosylribitol (PRP) polysaccharides extracted from Haemophilus influenzae type b (Hib), and the corresponding glycoconjugate made by conjugating this with the tetanus toxoid (TT) protein have been characterized and compared using a combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge with viscometry. The weight average molar mass of the activated material was considerably reduced (Mw ~ 0.24 × 10(6) g.mol(-1)) compared to the native (Mw ~ 1.2 × 10(6) g.mol(-1)). Conjugation with the TT protein yielded large polydisperse structures (of Mw ~ 7.4 × 10(6) g.mol(-1)), but which retained the high degree of flexibility of the native and activated polysaccharide, with frictional ratio, intrinsic viscosity, sedimentation conformation zoning behaviour and persistence length all commensurate with highly flexible coil behaviour and unlike the previously characterised tetanus toxoid protein (slightly extended and hydrodynamically compact structure with an aspect ratio of ~3). This non-protein like behaviour clearly indicates that it is the carbohydrate component which mainly influences the physical behaviour of the glycoconjugate in solution.

  20. Experimental design to optimize an Haemophilus influenzae type b conjugate vaccine made with hydrazide-derivatized tetanus toxoid.

    PubMed

    Laferriere, Craig; Ravenscroft, Neil; Wilson, Seanette; Combrink, Jill; Gordon, Lizelle; Petre, Jean

    2011-10-01

    The introduction of type b Haemophilus influenzae conjugate vaccines into routine vaccination schedules has significantly reduced the burden of this disease; however, widespread use in developing countries is constrained by vaccine costs, and there is a need for a simple and high-yielding manufacturing process. The vaccine is composed of purified capsular polysaccharide conjugated to an immunogenic carrier protein. To improve the yield and rate of the reductive amination conjugation reaction used to make this vaccine, some of the carboxyl groups of the carrier protein, tetanus toxoid, were modified to hydrazides, which are more reactive than the ε -amine of lysine. Other reaction parameters, including the ratio of the reactants, the size of the polysaccharide, the temperature and the salt concentration, were also investigated. Experimental design was used to minimize the number of experiments required to optimize all these parameters to obtain conjugate in high yield with target characteristics. It was found that increasing the reactant ratio and decreasing the size of the polysaccharide increased the polysaccharide:protein mass ratio in the product. Temperature and salt concentration did not improve this ratio. These results are consistent with a diffusion controlled rate limiting step in the conjugation reaction. Excessive modification of tetanus toxoid with hydrazide was correlated with reduced yield and lower free polysaccharide. This was attributed to a greater tendency for precipitation, possibly due to changes in the isoelectric point. Experimental design and multiple regression helped identify key parameters to control and thereby optimize this conjugation reaction.

  1. Long-term effects of tetanus toxoid inoculation on the demography and life expectancy of the Cayo Santiago rhesus macaques.

    PubMed

    Kessler, Matthew J; Hernández Pacheco, Raisa; Rawlins, Richard G; Ruiz-Lambrides, Angelina; Delgado, Diana L; Sabat, Alberto M

    2015-02-01

    Tetanus was a major cause of mortality in the free-ranging population of rhesus monkeys on Cayo Santiago prior to 1985 when the entire colony was given its first dose of tetanus toxoid. The immediate reduction in mortality that followed tetanus toxoid inoculation (TTI) has been documented, but the long-term demographic effects of eliminating tetanus infections have not. This study uses the Cayo Santiago demographic database to construct comparative life tables 12 years before, and 12 years after, TTI. Life tables and matrix projection models are used to test for differences in: (i) survival among all individuals as well as among social groups, (ii) long-term fitness of the population, (iii) age distribution, (iv) reproductive value, and (v) life expectancy. A retrospective life table response experiment (LTRE) was performed to determine which life cycle transition contributed most to observed changes in long-term fitness of the population post-TTI. Elimination of clinical tetanus infections through mass inoculation improved the health and well-being of the monkeys. It also profoundly affected the population by increasing survivorship and long-term fitness, decreasing the differences in survival rates among social groups, shifting the population's age distribution towards older individuals, and increasing reproductive value and life expectancy. These findings are significant because they demonstrate the long-term effects of eradicating a major cause of mortality at a single point in time on survival, reproduction, and overall demography of a naturalistic population of primates.

  2. Solution conformation and flexibility of capsular polysaccharides from Neisseria meningitidis and glycoconjugates with the tetanus toxoid protein

    PubMed Central

    Abdelhameed, Ali Saber; Morris, Gordon A.; Almutairi, Fahad; Adams, Gary G.; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E.

    2016-01-01

    The structural integrity of meningococcal native, micro-fluidized and activated capsular polysaccharides and their glycoconjugates – in the form most relevant to their potential use as vaccines (dilute solution) - have been investigated with respect to their homogeneity, conformation and flexibility. Sedimentation velocity analysis showed that the polysaccharide size distributions were generally bimodal with some evidence for higher molar mass forms at higher concentration. Weight average molar masses Mw where lower for activated polysaccharides. Conjugation with tetanus toxoid protein however greatly increased the molar mass and polydispersity of the final conjugates. Glycoconjugates had an approximately unimodal log-normal but broad and large molar mass profiles, confirmed by sedimentation equilibrium “SEDFIT MSTAR” analysis. Conformation analysis using HYDFIT (which globally combines sedimentation and viscosity data), “Conformation Zoning” and Wales-van Holde approaches showed a high degree of flexibility – at least as great as the unconjugated polysaccharides, and very different from the tetanus toxoid (TT) protein used for the conjugation. As with the recently published finding for Hib-TT complexes, it is the carbohydrate component that dictates the solution behaviour of these glycoconjugates, although the lower intrinsic viscosities suggest some degree of compaction of the carbohydrate chains around the protein. PMID:27782149

  3. An antibody induction method in mice for the potency assays of diphtheria and tetanus components in combined vaccines.

    PubMed

    Maheshwari, S C; Sharma, S B; Kumar, A; Sokhey, J

    1998-09-01

    Eleven batches of Adsorbed Diphtheria-Tetanus (DT) vaccines and thirteen batches of Adsorbed Diphtheria-Pertussis-Tetanus (DTP) vaccines were tested for the potency of diphtheria and tetanus components by an Antibody Induction Method (AIM) developed in mice. The potency results obtained were found comparable and did not show any statistically significant difference with those obtained by WHO recommended lethal challenge tests for diphtheria in guinea pigs and for tetanus in mice. AIM in mice is more economical as both diphtheria and tetanus components of combined vaccine can be tested in the same experiment and the procedure also eliminates the use of guinea pigs required in the lethal challenge/conventional tests. The data obtained while testing tetanus component by the conventional antibody induction (IP) method in guinea pigs suggests that minimum requirements laid down in i.p. is too low which may be fixed as at least 3 out of 9 guinea pig sera and should contain > or = 4 units of tetanus antitoxin per ml.

  4. Risk factors for diphtheria: a prospective case-control study in the Republic of Georgia, 1995-1996.

    PubMed

    Quick, M L; Sutter, R W; Kobaidze, K; Malakmadze, N; Nakashidze, R; Murvanidze, S; Wooten, K G; Strebel, P M

    2000-02-01

    The large-scale resurgence of diphtheria in the former Soviet Union offered a unique opportunity to evaluate risk factors for the transmission of respiratory diphtheria; therefore, a prospective case-control study was done in the republic of Georgia. In total, 218 diphtheria cases (hospitalized between October 1995 and March 1996) and 408 matched controls participated. One hundred cases (45%) were /=15 years of age (range: <1 to 75 years). In the multivariate analyses, the following risk factors were found to be significant: lack of vaccination (matched odds ratio [mOR]=19.2), household exposure to diphtheria (mOR=7.4), exposure to skin lesions (mOR=5.8), history of eczema (mOR=3.4), fever with myalgia prior to illness (mOR=2.6), having tonsils (mOR=4.4), sharing a bed (mOR=1.9), sharing cups and glasses (mOR=2.7), and taking a bath less than once a week (mOR=2.6). These findings emphasize primary prevention through immunizations, secondary prevention following exposure to diphtheria (and to suspicious skin lesions), and adherence to strict standards of personal hygiene.

  5. Protection of monkeys against the lethal effects of male funnel-web spider (Atrax robustus) venom by immunization with a toxoid.

    PubMed

    Sheumack, D D; Phillips, C A; Mylecharane, E J; Spence, I; Claassens, R; Brown, M R; Comis, A; Howden, M E

    1991-01-01

    A stable toxoid was prepared from robustoxin (the lethal polypeptide neurotoxin in the venom of the male funnel-web spider, Atrax robustus) by polymerization with glutaraldehyde. This material was non-toxic in new-born mice. Administration of the toxoid to three Macaca fascicularis monkeys (50-80 micrograms/kg s.c. at 14-day intervals for 8-12 weeks) produced no toxic effects; anti-robustoxin antibodies were detected in serum samples by immunodiffusion tests within 13-27 days. In vivo evidence of successful protection with the toxoid was obtained by challenging the monkeys with male A. robustus venom (50 micrograms/kg i.v.) under anaesthesia with pentobarbitone (one monkey), or with ketamine, halothane and nitrous oxide, 1-26 weeks after the last injection of the toxoid. Only minor respiratory, cardiovascular and skeletal motor disturbances were produced, and all monkeys recovered fully and uneventfully. Challenge with the same dose of venom in non-immunized or robustoxin N-terminal decapeptide ovalbumin conjugate-treated monkeys resulted in typical lethal neurotoxic effects, culminating in severe hypotension or death from circulatory and respiratory failure within 280 min.

  6. Synthesis of antifungal vaccines by conjugation of β-1,2 trimannosides with T-cell peptides and covalent anchoring of neoglycopeptide to tetanus toxoid.

    PubMed

    Cartmell, Jonathan; Paszkiewicz, Eugenia; Dziadek, Sebastian; Tam, Pui-Hang; Luu, Thanh; Sarkar, Susmita; Lipinski, Tomasz; Bundle, David R

    2015-02-11

    Selective strategies for the construction of novel three component glycoconjugate vaccines presenting Candida albicans cell wall glycan (β-1,2 mannoside) and polypeptide fragments on a tetanus toxoid carrier are described. The first of two conjugation strategies employed peptides bearing an N-terminal thiopropionyl residue for conjugation to a trisaccharide equipped with an acrylate linker and a C-terminal S-acetyl thioglycolyl moiety for subsequent linking of neoglycopeptide to bromoacetylated tetanus toxoid. Michael addition of acrylate trisaccharides to peptide thiol under mildly basic conditions gave a mixture of N- and C- terminal glyco-peptide thioethers. An adaptation of this strategy coordinated S-acyl protection with anticipated thioester exchange equilibria. This furnished a single chemically defined fully synthetic neoglycopeptide conjugate that could be anchored to a tetanus toxoid carrier and avoids the introduction of exogenous antigenic groups. The second strategy retained the N-terminal thiopropionyl residue but replaced the C-terminal S-acetate functionality with an azido group that allowed efficient, selective formation of neoglycopeptide thioethers and subsequent conjugation of these with propargylated tetanus toxoid, but introduced potentially antigenic triazole linkages.

  7. Meningococcal quadrivalent (serogroups A, C, W135 and Y) tetanus toxoid conjugate vaccine (Nimenrix™).

    PubMed

    Croxtall, Jamie D; Dhillon, Sohita

    2012-12-24

    Nimenrix™ (MenACWY-TT) is a quadrivalent meningococcal conjugate vaccine, comprising the polysaccharide serogroups A, C, W135 and Y, and tetanus toxoid (TT) as carrier protein. It is the first quadrivalent vaccine (administered as a single dose) to be approved in Europe for active immunization of individuals aged ≥ 12 months against invasive meningococcal disease caused by Neisseria meningitidis serogroups A, C, W135 and Y. Administration of a single dose of Nimenrix™ elicited a strong immune response against all four vaccine serogroups in healthy toddlers aged 12-23 months, children and adolescents aged 2-17 years and adults aged 18-55 years in randomized, multicentre, phase III trials. In toddlers, Nimenrix™ was noninferior to Meningitec® in terms of seroresponse rates against meningococcal serogroup C 42 days post-vaccination. In children, adolescents and adults, Nimenrix™ was noninferior to Mencevax™ in terms of vaccination response rates against all four serogroups 1 month post-vaccination. Furthermore, several phase II studies and a phase III trial showed that the immune response elicited by Nimenrix™ in all age groups persisted for 7-42 months after the primary vaccination (when evaluated by rabbit serum bactericidal activity), with the vaccine also inducing immune memory in toddlers. In addition, several randomized, multicentre, phase III, noninferiority trials showed that when coadministered with other childhood vaccines or a seasonal flu vaccine, the immunogenicity of Nimenrix™ or that of the coadministered vaccine was generally not altered. Nimenrix® was generally well tolerated in all age groups whether administered as a single vaccine or coadministered with other routine vaccines. The incidence of grade 3 local or systemic solicited adverse events during the first 4 days following vaccination and of serious adverse events over an extended follow-up period of up to 6 months was low (<4.5%). Although protective effectiveness and longer

  8. Immunogenicity and safety of combined adsorbed low-dose diphtheria, tetanus and inactivated poliovirus vaccine (REVAXIS®) versus combined diphtheria, tetanus and inactivated poliovirus vaccine (DT Polio®) given as a booster dose at 6 years of age

    PubMed Central

    Gajdos, Vincent; Soubeyrand, Benoit; Vidor, Emmanuel; Richard, Patrick; Boyer, Julie; Sadorge, Christine

    2011-01-01

    This randomized, comparative, phase-IIIb study conducted in France aimed to demonstrate whether seroprotection against diphtheria, tetanus and poliomyelitis 1 month after a single dose of REVAXIS (low-dose diphtheria) is non-inferior to seroprotection 1 month after a single dose of DT Polio (standard-dose diphtheria), both vaccines being given as a second booster to healthy children at 6 years of age. Children were randomly assigned to receive a single intramuscular dose of REVAXIS or DT Polio. Primary endpoints were the 1-month post-booster seroprotection rates for diphtheria, tetanus and poliovirus type-1, -2 and -3 antigens. Secondary endpoints were immunogenicity and safety observations. Of 788 children screened, 760 were randomized: REVAXIS group, 384 children; DT Polio group, 376 children. No relevant difference in demographic characteristics at baseline was observed between REVAXIS and DT Polio groups. Noninferiority of REVAXIS compared with DT Polio for seroprotection was demonstrated against diphtheria (respectively 98.6% and 99.3%), tetanus (respectively 99.6% and 100%) and poliovirus antigens (100% for each types in both groups). No allergic reactions to REVAXIS were reported. A benefit/risk ratio in favor of REVAXIS was suggested by the trend towards a better tolerability of REVAXIS compared with DT Polio regarding the rate of severe solicited injection-site reactions. The results support the use of REVAXIS as a booster at 6 years of age in infants who previously received a three-dose primary series within the first 6 months of life and a first booster including diphtheria, tetanus and poliovirus vaccine(s) given before 2 years of age. PMID:21441781

  9. Are we hitting immunity targets? The 2006 age-specific seroprevalence of measles, mumps, rubella, diphtheria and tetanus in Belgium.

    PubMed

    Theeten, H; Hutse, V; Hens, N; Yavuz, Y; Hoppenbrouwers, K; Beutels, P; Vranckx, R; Van Damme, P

    2011-04-01

    Susceptibility to vaccine-preventable diseases in Belgium in 2006 was estimated from a serum survey. Immunoglobulins against measles, mumps, rubella (MMR) and diphtheria at all available ages (1-65 years), and against tetanus in >40-year-olds, were measured by ELISA. Age-standardized overall seronegativity for MMR was low (3·9%, 8·0%, 10·4%, respectively). However, the World Health Organization's targets for measles elimination were not met in 5- to 24-year-olds and about 1 in 7 women at childbearing age (15-39 years) were seronegative for rubella. In adults >40 years, tetanus immunity (87·2%, >0·16 IU/ml) largely exceeded diphtheria immunity (20-45%, >0·1 IU/ml). Despite free universal vaccination against MMR for more than 20 years and against diphtheria and tetanus for almost 60 years, our study revealed specific age groups remaining at risk for infection with these pathogens.

  10. Novel potentiometry immunoassay with amplified sensitivity for diphtheria antigen based on Nafion, colloidal Ag and polyvinyl butyral as matrixes.

    PubMed

    Tang, Dianping; Yuan, Ruo; Chai, Yaqin; Zhang, Linyan; Zhong, Xia; Dai, Jianyuan; Liu, Yan

    2004-11-30

    A novel potentiometry immunoassay with amplified sensitivity has been developed for the detection of diphtheria antigen (Diph) via immobilizing diphtheria antibody (anti-Diph) on a platinum electrode based on Nafion, colloidal Ag (Ag), and polyvinyl butyral (PVB) as matrixes in this study. The modified procedure was further characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The influence and factors influencing the performance of resulting immunosensor were studied in detail. The resulting immunosensor exhibited sigmoid curve with log Diph concentrations, high sensitivity (51.4 mV/decade), wide linear range from 8 to 800 ng ml(-1) with a detection limit of 1.5 ng ml(-1), rapid potentiometric response (<3 min) and long-term stability (>6 months). Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting diphtheria antigen in the clinical diagnosis.

  11. Non-opsonic phagocytosis of homologous non-toxigenic and toxigenic Corynebacterium diphtheriae strains by human U-937 macrophages.

    PubMed

    dos Santos, Cíntia Silva; dos Santos, Louisy Sanches; de Souza, Monica Cristina; dos Santos Dourado, Fernanda; de Souza de Oliveira Dias, Alexandre Alves; Sabbadini, Priscila Soares; Pereira, Gabriela Andrade; Cabral, Maulori Curié; Hirata Junior, Raphael; de Mattos-Guaraldi, Ana Luíza

    2010-01-01

    As interactions between bacteria and macrophages dictate the outcome of most infectious diseases, analyses of molecular mechanisms of non-opsonic phagocytosis should lead to new approaches for the prevention of diphtheria and systemic Corynebacterium diphtheriae infections. The present study aimed to evaluate human macrophage-bacteria interactions in the absence of opsonin antibodies and the influence of the tox gene on this process. Homologous C. diphtheriae tox+ and tox- strains were evaluated for adhesion, entering and survival within U-937 human macrophages at different incubation periods. Higher numbers of viable bacteria associated with and internalized by macrophages were demonstrated for the tox+ strain. However, viable intracellular bacteria were detected at T-24 hr only for the tox- strain. Cytoskeletal inhibitors, cytochalasin E, genistein and colchicine, inhibited intracellular viability of both strains at different levels. Bacterial replication was evidenced at T-24 hr in supernatants of monolayers infected with the tox- strain. Host cell death and nuclear alterations were evidenced by the Trypan blue exclusion assay and DAPI fluorescence microscopy. ELISA of histone-associated DNA fragments allowed detection of apoptosis and necrosis induced by tox+ and tox- strains at T-1 hr and T-3 hr. In conclusion, human macrophages in the absence of opsonins may not be promptly effective at killing diphtheria bacilli. The presence of the tox gene influences the susceptibility of C. diphtheriae to human macrophages and the outcome of non-opsonic phagocytosis. C. diphtheriae strains exhibit strategies to survive within macrophages and to exert apoptosis and necrosis in human phagocytic cells, independent of the tox gene.

  12. Comparison of seven commercial enzyme-linked immunosorbent assays for the detection of anti-diphtheria toxin antibodies.

    PubMed

    Zasada, A A; Rastawicki, W; Śmietańska, K; Rokosz, N; Jagielski, M

    2013-07-01

    Determination of immune status of patients to diphtheria toxin is based mainly on the results of commercially available ELISA kits. The aim of the present study was to compare the results obtained by ELISAs from seven different manufacturers: Mikrogen, Immunolab, Sekisui Virotech, NovaTec, Virion\\Serion, IBL International and Euroimmun. All assays were performed according to the manufacturers' instructions. The concentrations of the anti-diphtheria toxin antibodies in 72 serum samples were calculated on the basis of curves constructed from standards supplied by manufacturers and the new reference material-International Standard for Diphtheria Antitoxin (10/262). The repeatability and reproducibility of all the ELISA kits tested were good. Number of sera with concentrations of the anti-diphtheria toxin antibodies below the WHO-recommended level of protection (0.1 IU/ml) were dependent on the ELISA used: Mikrogen, 20/72 samples (27.7%); Immunolab, 11/72 samples (15.3%); Sekisui Virotech, 0/72 samples (0%); NovaTec 18/72 samples (25.0%); Serion 12/72 samples (16.7%); IBL International, 7/72 samples (9.7 %); and Euroimmun, 17/72 samples (23.6%). However, the results obtained in particular ELISAs, with the exception of Sekisui Virotech, were much more consistent when the concentrations of the anti-diphtheria toxin antibodies in 72 sera measured by using curves constructed from the International Standard 10/262. The data obtained clearly demonstrated that manufacturer-dependent differences between anti-diphtheria IgG ELISA kits exist. The differences in recommendations accepted by the individual manufacturers together with differences shown in our studies in sensitivity greatly affect the clinical interpretation of results.

  13. Hepatitis B Vaccine

    MedlinePlus

    ... a combination product containing Haemophilus influenzae type b, Hepatitis B Vaccine) ... combination product containing Diphtheria, Tetanus Toxoids, Acellular Pertussis, Hepatitis B, Polio Vaccine)

  14. Corynebacterium diphtheriae as an emerging pathogen in nephrostomy catheter-related infection: evaluation of traits associated with bacterial virulence.

    PubMed

    Gomes, Débora L R; Martins, Carlos A S; Faria, Lúcia M D; Santos, Louisy S; Santos, Cintia S; Sabbadini, Priscila S; Souza, Mônica C; Alves, Gabriela B; Rosa, Ana C P; Nagao, Prescilla E; Pereira, Gabriela A; Hirata, Raphael; Mattos-Guaraldi, Ana L

    2009-11-01

    Corynebacterium diphtheriae still represents a global medical challenge, particularly due to the significant number of individuals susceptible to diphtheria and the emergence of non-toxigenic strains as the causative agents of invasive infections. In this study, we characterized the clinical and microbiological features of what we believe to be the first case of C. diphtheriae infection of a percutaneous nephrostomy catheter insertion site in an elderly patient with a fatal bladder cancer. Moreover, we demonstrated the potential role of adherence, biofilm formation and fibrin deposition traits in C. diphtheriae from the catheter-related infection. Non-toxigenic C. diphtheriae isolated from the purulent discharge (named strain BR-CAT5003748) was identified by the API Coryne system (code 1 010 324) and a multiplex PCR for detection of dtxR and tox genes. Strain BR-CAT5003748 showed resistance to oxacillin, ceftazidime and ciprofloxacin. In experiments performed in vitro, the catheter isolate was classified as moderately hydrophobic and as moderately adherent to polystyrene surfaces. Glass provided a more effective surface for biofilm formation than polystyrene. Micro-organisms adhered to (>1.5 x 10(6) c.f.u.) and multiplied on surfaces of polyurethane catheters. Microcolony formation (a hallmark of biofilm formation) and amorphous accretions were observed by scanning electron microscopy on both external and luminal catheter surfaces. Micro-organisms yielded simultaneous expression of localized adherence-like and aggregative-like (LAL/AAL) adherence patterns to HEp-2 cells. Interestingly, the coagulase tube test resulted in the formation of a thin layer of fibrin embedded in rabbit plasma by the non-toxigenic BR-CAT5003748 strain. In conclusion, C. diphtheriae should be recognized as a potential cause of catheter-related infections in at-risk populations such as elderly and cancer patients. LAL/AAL strains may be associated with virulence traits that enable C

  15. Channels Formed by Botulinum, Tetanus, and Diphtheria Toxins in Planar Lipid Bilayers: Relevance to Translocation of Proteins across Membranes

    NASA Astrophysics Data System (ADS)

    Hoch, David H.; Romero-Mira, Miryam; Ehrlich, Barbara E.; Finkelstein, Alan; Dasgupta, Bibhuti R.; Simpson, Lance L.

    1985-03-01

    The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as ``tunnel proteins'' for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.

  16. Microbiological and molecular characterization of Corynebacterium diphtheriae isolated in Algeria between 1992 and 2015.

    PubMed

    Benamrouche, N; Hasnaoui, S; Badell, E; Guettou, B; Lazri, M; Guiso, N; Rahal, K

    2016-12-01

    The objectives of this study were to undertake the microbiological and molecular characterization of Corynebacterium diphtheriae isolates collected in Algeria during epidemic and post-epidemic periods between 1992 and 2015. Microbiological characterization includes the determination of biotype and toxigenicity status using phenotypic and genotypic methods. Antimicrobial susceptibility was determined by the E-test method. Molecular characterization was performed by multi-locus sequence typing. In total, there were 157 cases of C. diphtheriae isolates, 127 in patients with respiratory diphtheria and 30 with ozena. Isolates with a mitis biotype were predominant (122 out of 157; 77.7%) followed by belfanti (28 out of 157; 17.8%) and gravis biotype (seven out of 157; 4.5%). Toxigenic isolates were predominant in the period 1992-2006 (74 out of 134) whereas in the period 2007-2015, only non-toxigenic isolates circulated (23 out of 23). All 157 isolates were susceptible to erythromycin, gentamicin, vancomycin and cotrimoxazole. Reduced susceptibility to penicillin G, cefotaxime, tetracycline and chloramphenicol was detected in 90 (57.3%), 88 (56.1%), 112 (71.3%) and 90 (57.3%) isolates, respectively. Multi-locus sequence typing analysis indicates that sequence type 116 (ST-116) was the most frequent, with 65 out of 100 isolates analysed, in particular during the epidemic period 1992-1999 (57 out of 65 isolates). In the post-epidemic period, 2000-2015, 13 different sequence types were isolated. All belfanti isolates (ten out of 100 isolates) belonged to closely related sequence types grouped in a phylogenetically distinct eBurst group and were collected exclusively in ozena cases. In conclusion, the epidemic period was associated with ST-116 while the post-epidemic period was characterized by more diversity. Belfanti isolates are grouped in a phylogenetically distinct clonal complex.

  17. Analyzing titers of antibodies against bacterial and viral antigens, and bacterial toxoids in the intravenous immunoglobulins utilized in Taiwan.

    PubMed

    Wu, Chi-Yu; Wang, Hsiu-Chi; Wang, Kun-Teng; Yang-Chih Shih, Daniel; Lo, Chi-Fang; Wang, Der-Yuan

    2013-03-01

    Intravenous immunoglobulin (IVIG) manufactured from human plasma contains IgG as the primary ingredient, and is used for indications such as immunodeficiency syndrome. Available IVIGs in Taiwan are either manufactured from Taiwanese or North American plasma. The effectiveness of the national immunization program of Taiwan can be evaluated by analyzing and comparing IVIG antibody titers that are induced through the corresponding vaccines (tetanus, diphtheria, and pertussis, measles, rubella, hepatitis A, hepatitis B and varicella). Both enzyme-linked immunosorbent assay (ELISA) and the in vitro neutralization test demonstrated that all IVIGs provide adequate clinical protection against diphtheria and tetanus toxins. ELISA results further revealed that plasma of Taiwanese subjects contains higher levels of pertussis toxin and filamentous hemagglutinin antibodies, when compared to foreign IVIGs. This may be related to the later adoption of acellular pertussis vaccine in Taiwan. Antibodies titers against measles, rubella, hepatitis A, and varicella-zoster virus were otherwise low. Low titers of hepatitis B surface antigen antibodies are present in Taiwanese plasma IVIG, indicating immune memory decline or loss. In conclusion, our results show that Taiwanese IVIG contains varying titers of vaccine-induced antibodies, and serves as a guide for future amendments to Taiwan's immunization program.

  18. [Preparation of cationic dextran microspheres loaded with tetanus toxoid and study on the mechanism of protein loading].

    PubMed

    Zheng, Chun-li; Liu, Xiao-qing; Zhu, Jia-bi; Zhao, Yu-na

    2010-09-01

    The aim of this study is to prepare cationic biodegradable dextran microspheres loaded with tetanus toxoid (TT) and to investigate the mechanism of protein loading. Positively charged microspheres were prepared by polymerization of hydroxylethyl methacrylate derivatized dextran (dex-HEMA) and dimethyl aminoethyl methacrylate (DMAEMA) in an aqueous two-phase system. The loading of the microspheres with TT was based on electrostatic attraction. The net positive surface charge increased with increasing amounts of DMAEMA. Confocal images showed fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) could penetrate into cationic dextran microspheres but not natural dextran microspheres. TT loading efficiency by post-loading was higher compared with by pre-loading. Even though TT is incorporated in the hydrogel network based on electrostatic interaction, still a controlled release can be achieved by varying the initial network density of the microspheres.

  19. Support Between USAMRDC and USDA for Cooperative Research Under the Ration Sustainment Testing Program.

    DTIC Science & Technology

    1992-12-31

    filtrate from the following microorganisms: Clostridium tetani (tetanus toxoid), Corynebacterium diphtheriae ( diphtheria toxoid), StrepDtococcus, Group C...responses to test antigens diphtheria , at the end of the jungle phase, proteus, at the end of both the jungle and desert phases, and tuberculin, at the end of...baseline and at the end of jungle and desert phases. Antigen Baseline Jungle Desert Tetanus 91 87 85 Diphtheria 56 a 74 b 6 5a,b Candlda 56 56 56

  20. Emergence and molecular characterisation of non-toxigenic tox gene-bearing Corynebacterium diphtheriae biovar mitis in the United Kingdom, 2003-2012.

    PubMed

    Zakikhany, K; Neal, S; Efstratiou, A

    2014-06-05

    Non-toxigenic Corynebacterium diphtheriae have become increasingly recognised as emerging pathogens across Europe causing severe invasive disease. A subset of non-toxigenic C. diphtheriae are ‘non-toxigenic tox gene-bearing’ (NTTB) strains; these strains are genotypically toxpositive, but do not express the protein. The circulation of NTTB strains was first observed during the 1990s upsurge of diphtheria in Eastern Europe but has not been reported in other European countries. Circulation of NTTB strains could be considered an increased risk for diphtheria and other related diseases, given their possible role as a tox gene reservoir with the theoretical risk of re-emerging toxin expression. Here we report the characterisation of 108 non-toxigenic C. diphtheriae biovar mitis isolates submitted to the World Health Organization (WHO) Global Reference Centre for Diphtheria at Public Health England, London, between 2003 and 2012, in order to determine the presence of NTTB strains. Using molecular methods, five NTTB isolates were identified; four human isolates (MLST type 212) and one isolate from a companion cat (MLST type 40). The emergence of these strains could indicate continuation of the circulation of potentially toxigenic strains and appropriate laboratory diagnostic methods should be used for detection. Given the complacency that currently exists in Europe awareness with regards to diphtheria diagnostics must be enhanced.

  1. A brain-sparing diphtheria toxin for chemical genetic ablation of peripheral cell lineages.

    PubMed

    Pereira, Mafalda M A; Mahú, Inês; Seixas, Elsa; Martinéz-Sánchez, Noelia; Kubasova, Nadiya; Pirzgalska, Roksana M; Cohen, Paul; Dietrich, Marcelo O; López, Miguel; Bernardes, Gonçalo J L; Domingos, Ana I

    2017-04-03

    Conditional expression of diphtheria toxin receptor (DTR) is widely used for tissue-specific ablation of cells. However, diphtheria toxin (DT) crosses the blood-brain barrier, which limits its utility for ablating peripheral cells using Cre drivers that are also expressed in the central nervous system (CNS). Here we report the development of a brain-sparing DT, termed BRAINSPAReDT, for tissue-specific genetic ablation of cells outside the CNS. We prevent blood-brain barrier passage of DT through PEGylation, which polarizes the molecule and increases its size. We validate BRAINSPAReDT with regional genetic sympathectomy: BRAINSPAReDT ablates peripheral but not central catecholaminergic neurons, thus avoiding the Parkinson-like phenotype associated with full dopaminergic depletion. Regional sympathectomy compromises adipose tissue thermogenesis, and renders mice susceptible to obesity. We provide a proof of principle that BRAINSPAReDT can be used for Cre/DTR tissue-specific ablation outside the brain using CNS drivers, while consolidating the link between adiposity and the sympathetic nervous system.

  2. Impaired cellular immune response to diphtheria and tetanus vaccines in children after thoracic transplantation.

    PubMed

    Urschel, Simon; Rieck, Birgit D; Birnbaum, Julia; Dalla Pozza, Robert; Rieber, Nikolaus; Januszewska, Katarzyna; Fuchs, Alexandra; West, Lori J; Netz, Heinrich; Belohradsky, Bernd H

    2011-05-01

    Safety and immunogenicity of diphtheria and tetanus booster vaccination were evaluated in 28 children after thoracic transplantation. Adverse events were documented in a patient diary. Blood was collected prior to and four wk after vaccination. Specific antibody concentrations were measured by ELISA. Lymphocytes were investigated for expression of activation markers (CD25, HLA-DR) by flow cytometry and proliferation assays with and without stimulation. Post-vaccination antibody titers were higher than prevaccination (p < 0.001), with more patients having protective antibody levels against diphtheria (p < 0.02) and tetanus (p < 0.001). There was no increased proliferation in non-stimulated or stimulated cultures after vaccination. The number of T-lymphocytes activated by the vaccination antigens was similar pre- and post-vaccination, whereas HLA-DR-expression on stimulated and non-stimulated CD4(+) T-cells increased significantly. Increase in antibodies was negatively correlated with tacrolimus dose, and impaired cellular immunity was associated with higher tacrolimus dose and steroid use. Adverse events were similar to the general population; serious adverse events and rejection did not occur. Vaccination with inactivated vaccines can be performed safely in immunosuppressed children after thoracic transplantation and induces protective antibody levels in the majority of patients. Impaired induction of specific cellular immunity is correlated with intensity of immunosuppression and may explain reduced sustainability of antibodies.

  3. Depletion of alveolar macrophages in CD11c diphtheria toxin receptor mice produces an inflammatory response.

    PubMed

    Roberts, Lydia M; Ledvina, Hannah E; Tuladhar, Shraddha; Rana, Deepa; Steele, Shaun P; Sempowski, Gregory D; Frelinger, Jeffrey A

    2015-06-01

    Alveolar macrophages play a critical role in initiating the immune response to inhaled pathogens and have been shown to be the first cell type infected following intranasal inoculation with several pathogens, including Francisella tularensis. In an attempt to further dissect the role of alveolar macrophages in the immune response to Francisella, we selectively depleted alveolar macrophages using CD11c.DOG mice. CD11c.DOG mice express the diphtheria toxin receptor (DTR) under control of the full CD11c promoter. Because mice do not express DTR, tissue restricted expression of the primate DTR followed by treatment with diphtheria toxin (DT) has been widely used as a tool in immunology to examine the effect of acute depletion of a specific immune subset following normal development. We successfully depleted alveolar macrophages via intranasal administration of DT. However, alveolar macrophage depletion was accompanied by many other changes to the cellular composition and cytokine/chemokine milieu in the lung that potentially impact innate and adaptive immune responses. Importantly, we observed a transient influx of neutrophils in the lung and spleen. Our experience serves as a cautionary note to other researchers using DTR mice given the complex changes that occur following DT treatment that must be taken into account when analyzing data.

  4. Depletion of alveolar macrophages in CD11c diphtheria toxin receptor mice produces an inflammatory response

    PubMed Central

    Roberts, Lydia M; Ledvina, Hannah E; Tuladhar, Shraddha; Rana, Deepa; Steele, Shaun P; Sempowski, Gregory D; Frelinger, Jeffrey A

    2015-01-01

    Alveolar macrophages play a critical role in initiating the immune response to inhaled pathogens and have been shown to be the first cell type infected following intranasal inoculation with several pathogens, including Francisella tularensis. In an attempt to further dissect the role of alveolar macrophages in the immune response to Francisella, we selectively depleted alveolar macrophages using CD11c.DOG mice. CD11c.DOG mice express the diphtheria toxin receptor (DTR) under control of the full CD11c promoter. Because mice do not express DTR, tissue restricted expression of the primate DTR followed by treatment with diphtheria toxin (DT) has been widely used as a tool in immunology to examine the effect of acute depletion of a specific immune subset following normal development. We successfully depleted alveolar macrophages via intranasal administration of DT. However, alveolar macrophage depletion was accompanied by many other changes to the cellular composition and cytokine/chemokine milieu in the lung that potentially impact innate and adaptive immune responses. Importantly, we observed a transient influx of neutrophils in the lung and spleen. Our experience serves as a cautionary note to other researchers using DTR mice given the complex changes that occur following DT treatment that must be taken into account when analyzing data. PMID:26029367

  5. Dmp1 Promoter-Driven Diphtheria Toxin Receptor Transgene Expression Directs Unforeseen Effects in Multiple Tissues

    PubMed Central

    Al-Jazzar, Ahmed; Javaheri, Behzad; Prideaux, Matt; Boyde, Alan; Scudamore, Cheryl L.; Cherifi, Chahrazad; Hay, Eric; Hopkinson, Mark; Boyd, Michael; Cohen-Solal, Martine; Farquharson, Colin; Pitsillides, Andrew A.

    2016-01-01

    Mice harbouring a dentin matrix protein 1 (Dmp1) promoter-driven human diphtheria toxin (DT) receptor (HDTR) transgene (Tg) have recently been used to attain targeted ablation of osteocytes by diphtheria toxin (DT) treatment in order to define osteocyte function. Use of these Tg mice has asserted mechano- and novel paracrine regulatory osteocyte functions. To explore osteocyte roles fully, we sought to confirm the selectivity of DT effects in these transgenic mice. However, our findings revealed incomplete DT-induced osteocyte ablation, prevalent HDTR misexpression, as well as more prominent histopathological DT-induced changes in multiple organs in Tg than in wild-type (WT) littermate mice. Mechanistic evidence for DT action, via prominent regulation of phosphorylation status of elongation factor-2 (EF-2), was also found in many non-skeletal tissues in Tg mice; indicative of direct “off-target” DT action. Finally, very rapid deterioration in health and welfare status in response to DT treatment was observed in these Tg when compared to WT control mice. Together, these data lead us to conclude that alternative models for osteocyte ablation should be sought and caution be exercised when drawing conclusions from experiments using these Tg mice alone. PMID:28035954

  6. Immunogenicity and reactogenicity of the adult tetanus-diphtheria vaccine. How many doses are necessary?

    PubMed Central

    Bayas, J. M.; Vilella, A.; Bertran, M. J.; Vidal, J.; Batalla, J.; Asenjo, M. A.; Salleras, L. L.

    2001-01-01

    The immunogenicity and reactogenicity of the tetanus-diphtheria adult type vaccine was compared in two groups: group I (n = 201, 18-30 years old, presumably vaccinated with the DTP vaccine) and group II (n = 147, > or = 45 years old, without vaccination antecedents). Before vaccination, the seroprotection levels for tetanus were 90.5% (group I) and 30.6% (group II). These rose to 99.5% and 81.7%, respectively, after administration of one vaccine dose. For diphtheria, prevaccination seroprotection levels were 38.3% (group I) and 19.0% (group II). These rose to 85.8% and 65.7%, respectively, after vaccination. The logistic regression analysis showed an association between antibody titre and age. In group II, 3 doses of Td vaccine were needed to reach titres similar to those achieved in group I with a single dose. Stated reactogenicity was greater in: young subjects, women, those with higher titres of tetanus antibodies and those receiving other vaccines simultaneously. These results confirm the need for vaccination schedules adapted to the characteristics of each population age-group. PMID:11811878

  7. Differential Chemical Protection of Mammalian Cells from the Exotoxins of ’Corynebacterium diphtheriae’ and ’Pseudomonas aeruginosa’,

    DTIC Science & Technology

    Many drugs or chemicals had markedly different effects on the cytotoxicity induced by Pseudomonas aeruginosa exotoxin A (PE) or Corynebacterium ... diphtheriae exotoxin (DE). The glycolytic inhibitor NaF protected cells from DE but potentiated the cytotoxicity of PE. Another energy inhibitor, salicylic

  8. No influence of atopic diseases on antibody titres following tetanus, diphtheria and hepatitis B immunisation among adults.

    PubMed

    Friedrich, N; Kramer, A; Mentel, R; Gürtler, L; John, U; Völzke, H

    2007-12-01

    Several studies have reported associations between reduced humoral immune response to vaccine antigens and diseases with modified reactions of the immune system. We have investigated the influence of atopic diseases on specific IgG levels to tetanus, diphtheria and hepatitis B (HB), following immunisation, in a general adult population. From the Study of Health in Pomerania, a total number of 3,920 subjects aged 20 to 79 years were included in the analyses. Information on immunisation history, as well as behavioural and socio-demographic characteristics were collected. Anti-tetanus IgG, anti-diphtheria IgG and anti-HBs IgG were measured by indirect enzyme-linked immunosorbent assay (ELISA). Odds ratios and 95% confidence intervals were calculated using logistic regression. Atopic diseases were reported by 14% of participants. Proportions of 67%, 34% and 10% had been vaccinated against tetanus, diphtheria and hepatitis B within the past ten years, respectively. Multi-variable analyses revealed no associations between the presence of atopic diseases and all of the three vaccine-specific antibody titres. We conclude that there is no reduced immune response related to antibody production following immunisations against tetanus, diphtheria and hepatitis B in adults with atopic diseases.

  9. Long-Term Protection against Diphtheria in the Netherlands after 50 Years of Vaccination: Results from a Seroepidemiological Study

    PubMed Central

    Swart, E. M.; van Gageldonk, P. G. M.; de Melker, H. E.; van der Klis, F. R.; Berbers, G. A. M.; Mollema, L.

    2016-01-01

    Background and Aims To evaluate the National Immunisation Programme (NIP) a population-based cross-sectional seroepidemiological study was performed in the Netherlands. We assessed diphtheria antitoxin levels in the general Dutch population and in low vaccination coverage (LVC) areas where a relatively high proportion of orthodox Protestants live who decline vaccination based on religious grounds. Results were compared with a nationwide seroepidemiological study performed 11 years earlier. Methods In 2006/2007 a national serum bank was established. Blood samples were tested for diphtheria antitoxin IgG concentrations using a multiplex immunoassay for 6383 participants from the national sample (NS) and 1518 participants from LVC municipalities. A cut-off above 0.01 international units per ml (IU/ml) was used as minimum protective level. Results In the NS 91% of the population had antibody levels above 0.01 IU/ml compared to 88% in the 1995/1996 serosurvey (p<0.05). On average, 82% (vs. 78% in the 1995/1996 serosurvey, p<0.05) of individuals from the NS born before introduction of diphtheria vaccination in the NIP and 46% (vs. 37% in the 1995/1996 serosurvey, p = 0.11) of orthodox Protestants living in LVC areas had antibody levels above 0.01 IU/ml. Linear regression analysis among fully immunized individuals (six vaccinations) without evidence of revaccination indicated a continuous decline in antibodies in both serosurveys, but geometric mean antibodies remained well above 0.01 IU/ml in all age groups. Conclusions The NIP provides long-term protection against diphtheria, although antibody levels decline after vaccination. As a result of natural waning immunity, a substantial proportion of individuals born before introduction of diphtheria vaccination in the NIP lack adequate levels of diphtheria antibodies. Susceptibility due to lack of vaccination is highest among strictly orthodox Protestants. The potential risk of spread of diphtheria within the geographically

  10. Antibody Persistence in Young Children 5 Years after Vaccination with a Combined Haemophilus influenzae Type b-Neisseria meningitidis Serogroup C Conjugate Vaccine Coadministered with Diphtheria-Tetanus-Acellular Pertussis-Based and Pneumococcal Conjugate Vaccines.

    PubMed

    Tejedor, Juan Carlos; Brzostek, Jerzy; Konior, Ryszard; Grunert, Detlef; Kolhe, Devayani; Baine, Yaela; Van Der Wielen, Marie

    2016-07-01

    We evaluated antibody persistence in children up to 5 years after administration of a combined Haemophilus influenzae type b (Hib)-Neisseria meningitidis serogroup C (MenC)-tetanus toxoid (TT) conjugate vaccine coadministered with a pneumococcal conjugate vaccine. This is the follow-up study of a randomized trial (ClinicalTrials.gov registration no. NCT00334334/00463437) in which healthy children were vaccinated (primary vaccinations at 2, 4, and 6 months of age and booster vaccination at 11 to 18 months of age) with Hib-MenC-TT or a control MenC conjugate vaccine, coadministered with diphtheria-tetanus-acellular pertussis (DTPa)-based combination vaccines (DTPa/Hib for control groups) and a pneumococcal conjugate vaccine (10-valent pneumococcal nontypeable H. influenzae protein D conjugate vaccine [PHiD-CV] or 7-valent cross-reacting material 197 [CRM197] conjugate vaccine [7vCRM]). MenC antibody titers were measured with a serum bactericidal antibody (SBA) assay using rabbit complement (i.e., rabbit SBA [rSBA]), and antibodies against Hib polyribosylribitol phosphate (PRP) were measured with an enzyme-linked immunosorbent assay. Antibody persistence up to 5 years after booster vaccination is reported for 530 children ∼6 years of age. The percentages of children with seroprotective rSBA-MenC titers were between 24.2% and 40.1% in all groups approximately 5 years after booster vaccination. More than 98.5% of children in each group retained seroprotective anti-PRP concentrations. No vaccine-related serious adverse events and no events related to a lack of vaccine efficacy were reported. Approximately 5 years after booster vaccination, the majority of children retained seroprotective anti-PRP antibody concentrations. The percentage of children retaining seroprotective rSBA-MenC titers was low (≤40%), suggesting that a significant proportion of children may be unprotected against MenC disease. (This study has been registered at ClinicalTrials.gov under

  11. Antibody Persistence in Young Children 5 Years after Vaccination with a Combined Haemophilus influenzae Type b-Neisseria meningitidis Serogroup C Conjugate Vaccine Coadministered with Diphtheria-Tetanus-Acellular Pertussis-Based and Pneumococcal Conjugate Vaccines

    PubMed Central

    Tejedor, Juan Carlos; Brzostek, Jerzy; Konior, Ryszard; Grunert, Detlef; Kolhe, Devayani; Baine, Yaela

    2016-01-01

    We evaluated antibody persistence in children up to 5 years after administration of a combined Haemophilus influenzae type b (Hib)-Neisseria meningitidis serogroup C (MenC)-tetanus toxoid (TT) conjugate vaccine coadministered with a pneumococcal conjugate vaccine. This is the follow-up study of a randomized trial (ClinicalTrials.gov registration no. NCT00334334/00463437) in which healthy children were vaccinated (primary vaccinations at 2, 4, and 6 months of age and booster vaccination at 11 to 18 months of age) with Hib-MenC-TT or a control MenC conjugate vaccine, coadministered with diphtheria-tetanus-acellular pertussis (DTPa)-based combination vaccines (DTPa/Hib for control groups) and a pneumococcal conjugate vaccine (10-valent pneumococcal nontypeable H. influenzae protein D conjugate vaccine [PHiD-CV] or 7-valent cross-reacting material 197 [CRM197] conjugate vaccine [7vCRM]). MenC antibody titers were measured with a serum bactericidal antibody (SBA) assay using rabbit complement (i.e., rabbit SBA [rSBA]), and antibodies against Hib polyribosylribitol phosphate (PRP) were measured with an enzyme-linked immunosorbent assay. Antibody persistence up to 5 years after booster vaccination is reported for 530 children ∼6 years of age. The percentages of children with seroprotective rSBA-MenC titers were between 24.2% and 40.1% in all groups approximately 5 years after booster vaccination. More than 98.5% of children in each group retained seroprotective anti-PRP concentrations. No vaccine-related serious adverse events and no events related to a lack of vaccine efficacy were reported. Approximately 5 years after booster vaccination, the majority of children retained seroprotective anti-PRP antibody concentrations. The percentage of children retaining seroprotective rSBA-MenC titers was low (≤40%), suggesting that a significant proportion of children may be unprotected against MenC disease. (This study has been registered at ClinicalTrials.gov under

  12. Diphtheria toxin IgG levels in military and civilian blood donors in Rio de Janeiro, Brazil.

    PubMed

    Speranza, F A B; Ishii, S K; Hirata, R; Mattos-Guaraldi, A L; Milagres, L G

    2010-01-01

    Serologic data on diseases that are preventable by vaccines are necessary to evaluate the success of immunization programs and to identify susceptible subgroups. In the present study, we determined serum IgG levels against diphtheria toxin of military and civilian blood donors (N = 75; 69.3% males and 30.7% females) aged 18-64 years, from the Brazilian Army Biology Institute, Rio de Janeiro, using a commercial diphtheria kit (Diphtheria IgG ELISA; IBL, Germany). Most (63%) unprotected military donors were from the older age group of 41 to 64 years. In contrast, the majority (71%) of young military donors (18 to 30 years) were fully protected. About half of the military donors aged 31 to 40 years were protected against diphtheria. Among the civilians, about 50% of persons aged 18 to 30 years and 31 to 40 years had protective antibody levels against diphtheria as also did 64% of individuals aged 41 to 64 years. All civilians had a similar antibody response (geometric mean = 0.55 IU/mL) independent of age group. Military donors aged 18-30 years had higher IgG levels (geometric mean = 0.82 IU/mL) than military donors of 41-64 years (geometric mean = 0.51 IU/mL; P > 0.05). In conclusion, the existence of a considerable proportion of susceptible adults supports the position that reliable data on the immune status of the population should be maintained routinely and emphasizes the importance of adequate immunization during adulthood.

  13. Scaling up interventions to eliminate neonatal tetanus: factors associated with the coverage of tetanus toxoid and clean deliveries among women in Vientiane, Lao PDR.

    PubMed

    Masuno, Kanako; Xaysomphoo, Duangpachan; Phengsavanh, Alongkone; Douangmala, Somthana; Kuroiwa, Chushi

    2009-07-09

    The Lao People's Democratic Republic (PDR) is one of seven countries that have not eliminated maternal and neonatal tetanus in more than 50% of districts. We conducted a community-based household survey to assess the achievements of strategies towards maternal and neonatal tetanus elimination in the capital province. The coverage of tetanus toxoid (TT) was 79.7% by the protection-at-birth (PAB) method. The percentages of deliveries attended by skilled personnel and of deliveries at a health facility were 68.4% and 63.7%, respectively. The progress towards eliminating neonatal tetanus in Lao PDR is not sufficient despite the study sites being placed in the capital province. The lack of continuum of care for mothers and newborns is the major obstacle to scale up the tetanus toxoid coverage and PAB as well as clean deliveries.

  14. Comparison of three enzyme-linked immunosorbent assays for detection of immunoglobulin g antibodies to tetanus toxoid with reference standards and the impact on clinical practice.

    PubMed

    van Hoeven, Karen H; Dale, Connie; Foster, Phil; Body, Barbara

    2008-12-01

    Accurate determination of the concentrations of immunoglobulin G (IgG) antibody to tetanus toxoid is important in order to evaluate the immunogenicity of tetanus toxoid vaccines, determine immune competence in individual patients, and measure the prevalence of immunity in populations. The performance of three commercially available enzyme-linked immunosorbent assays (ELISAs) for IgG antibodies to tetanus toxoid were evaluated. Serially diluted NIBSC 76/589 and TE-3 human tetanus IgG immunoglobulin international reference standards were analyzed in quadruplicate using ELISAs manufactured by The Binding Site, Inc. (VaccZyme); Scimedx; and Euroimmun. In addition, IgG antibodies to tetanus toxoid were measured in 83 deidentified serum specimens using each manufacturer's ELISA. Each ELISA provided linear results when evaluated with the reference preparations. The Binding Site ELISA provided results that closely corresponded to the reference preparations (y=1.09x-0.08), whereas the Scimedx ELISA gave results that were consistently lower (y=0.21x-0.07) and the Euroimmun ELISA gave results that were consistently higher (y=1.5x+0.30) than the reference preparation concentrations. Using the recommended cutoff for each ELISA (<0.10 IU/ml), the overall agreement of all of the ELISA methods was 78%. Three of eighty-three (3.6%) human serum samples demonstrated inadequate immunity with all three assays. The Binding Site ELISA yielded nonprotective antibody concentrations in only these 3 samples, whereas 19 samples (22.9%) according to the Scimedx ELISA and 6 samples (7.2%) according to the Euroimmun ELISA demonstrated nonprotective concentrations. The performance characteristics of ELISAs for tetanus immunoglobulin titers were manufacturer dependent, and the differences translated into important disparities in reported results.

  15. Vaccination with parenteral toxoid B protects hamsters against lethal challenge with toxin A-negative, toxin B-positive clostridium difficile but does not prevent colonization.

    PubMed

    Siddiqui, Farida; O'Connor, Jennifer R; Nagaro, Kristin; Cheknis, Adam; Sambol, Susan P; Vedantam, Gayatri; Gerding, Dale N; Johnson, Stuart

    2012-01-01

    Toxin A has historically been regarded as the primary virulence determinant in Clostridium difficile infection, but naturally occurring toxin A-negative, toxin B-positive (A-/B+) C. difficile strains are known to be virulent. To determine the role of toxin B in these strains, we immunized hamsters with a toxoid prepared from purified toxin B to determine whether they would be protected from lethal challenge with an A-/B+ strain of C. difficile.

  16. Immunogenicity and efficacy against lethal aerosol staphylococcal enterotoxin B challenge in monkeys by intramuscular and respiratory delivery of proteosome-toxoid vaccines.

    PubMed Central

    Lowell, G H; Colleton, C; Frost, D; Kaminski, R W; Hughes, M; Hatch, J; Hooper, C; Estep, J; Pitt, L; Topper, M; Hunt, R E; Baker, W; Baze, W B

    1996-01-01

    Staphylococcal enterotoxin B (SEB), a primary cause of food poisoning, is also a superantigen that can cause toxic shock after traumatic or surgical staphylococcal wound [correction of would] infections or viral influenza-associated staphylococcal superinfections or when aerosolized for use as a potential biologic warfare threat agent. Intranasal or intramuscular (i.m.) immunization with formalinized SEB toxoid formulated with meningococcal outer membrane protein proteosomes has previously been shown to be immunogenic and protective against lethal respiratory or parenteral SEB challenge in murine models of SEB intoxication. Here, it is demonstrated that immunization of nonhuman primates with the proteosome-SEB toxoid vaccine is safe, immunogenic, and protective against lethal aerosol challenge with 15 50% lethal doses of SEB. Monkeys (10 per group) were primed i.m. and given booster injections by either the i.m. or intratracheal route without adverse side effects. Anamnestic anti-SEB serum immunoglobulin G (IgG) responses were elicited in all monkeys, but strong IgA responses in sera and bronchial secretions were elicited both pre- and post-SEB challenge only in monkeys given booster injections intratracheally. The proteosome-SEB toxoid vaccine was efficacious by both routes in protecting 100% of monkeys against severe symptomatology and death from aerosolized-SEB intoxication. These data confirm the safety, immunogenicity, and efficacy in monkeys of parenteral and respiratory vaccination with the proteosome-SEB toxoid, thereby supporting clinical trials of this vaccine in humans. The safety and enhancement of both bronchial and systemic IgA and IgG responses by the proteosome vaccine delivered by a respiratory route are also encouraging for the development of mucosally delivered proteosome vaccines to protect against SEB and other toxic or infectious respiratory pathogens. PMID:8890226

  17. Screening for Corynebacterium diphtheriae and Corynebacterium ulcerans in patients with upper respiratory tract infections 2007-2008: a multicentre European study.

    PubMed

    Wagner, K S; White, J M; Neal, S; Crowcroft, N S; Kuprevičiene, N; Paberza, R; Lucenko, I; Jõks, U; Akbaş, E; Alexandrou-Athanassoulis, H; Detcheva, A; Vuopio, J; von Hunolstein, C; Murphy, P G; Andrews, N; Efstratiou, A

    2011-04-01

    Diphtheria is now rare in most European countries but, when cases do arise, the case fatality rate is high (5-10%). Because few countries continue to routinely screen for the causative organisms of diphtheria, the extent to which they are circulating amongst different European populations is largely unknown. During 2007-2008, ten European countries each screened between 968 and 8551 throat swabs from patients with upper respiratory tract infections. Six toxigenic strains of Corynebacterium diphtheriae were identified: two from symptomatic patients in Latvia (the country with the highest reported incidence of diphtheria in the European Union) and four from Lithuania (two cases, two carriers); the last reported case of diphtheria in Lithuania was in 2002. Carriage rates of non-toxigenic organisms ranged from 0 (Bulgaria, Finland, Greece, Ireland, Italy) to 4.0 per 1000 (95% CI 2.0-7.1) in Turkey. A total of 28 non-toxigenic strains were identified during the study (26 C. diphtheriae, one Corynebacterium ulcerans, one Corynebacterium pseudotuberculosis). The non-toxigenic C. ulcerans strain was isolated from the UK, the country with the highest reported incidence of cases due to C. ulcerans. Of the eleven ribotypes detected, Cluj was seen most frequently in the non-toxigenic isolates and, amongst toxigenic isolates, the major epidemic clone, Sankt-Petersburg, is still in circulation. Isolation of toxigenic C. diphtheriae and non-toxigenic C. diphtheriae and C. ulcerans in highly-vaccinated populations highlights the need to maintain microbiological surveillance, laboratory expertise and an awareness of these organisms amongst public health specialists, microbiologists and clinicians.

  18. [Literature memories about diphtheria: Mark Twain, W.G. Sebald and Stendhal syndrome].

    PubMed

    Ledermann, Walter

    2013-02-01

    Memories of W.G. Sebald from the diphtheria he suffered as a child, gave rise to a discussion about the origin of classic clinical descriptions and the traps memory tends. Good examples of the latter are some experiences of Stendhal, who must also be distrusted given his hypersensitivity, which gave name to a psychosomatic syndrome. Mark Twain, a more practical man, brings us back to reality with a funny story about the terror the disease caused in the late nineteenth century. This leads us to remember isolation measures and topical treatments from the period immediately preceding the antitoxin. They included manual removal of the pseudo membranes, maneuver that led Marañón to misinterpret a painting by Goya on a scene of "Lazarillo de Tormes".

  19. Bone erosion and subacromial bursitis caused by diphtheria-tetanus-poliomyelitis vaccine.

    PubMed

    Salmon, J H; Geoffroy, M; Eschard, J P; Ohl, X

    2015-11-17

    Revaxis(®) is a vaccine against diphtheria, tetanus and poliomyelitis (dT-IPV). This vaccine should not be administered by the intradermal or intravenous route. Poor injection techniques and related consequences are rare. We report a case of bursitis associated with reactive glenohumeral effusion complicated by bone erosion occurring after injection of the dT-IPV vaccine. A 26 year old patient was admitted for painful left shoulder causing functional impairment. Control magnetic resonance imaging showed bone oedema on the upper outer part of the humeral head, with a slight cortical irregularity, indicating that the vaccine was injected in contact with the bone at this location, causing erosion. Outcome was favourable after intra-articular corticosteroids. Reports of articular or periarticular injury after vaccination are extremely rare, in view of the substantial number of vaccines administered every year. The potential complications of vaccination are well known to general practitioners but under-reported in the literature.

  20. Diphthericin types, bacteriophage types and serotypes of Corynebacterium diphtheriae strains isolated in Australia

    PubMed Central

    Gibson, L. F.; Colman, G.

    1973-01-01

    A dipthericin typing scheme has been constructed using 441 strains of Corynebacterium diptheriae isolated in eastern Australia from 1962 to 1971. Ten types have been distinguished using seven strains of C. diphtheriae and two strains of C. belfanti as indicators of the diphthericins produced by the newly isolated strains. Strains grouped into types L2, L3 and L3a were found only in Melbourne and types L1 and L4 were predominant in Sydney. Type L5 strains were isolated intermittently throughout the period of study and were found in all eastern states. Numerical analysis of the characteristics of the strains suggests that associations exist between, on the one hand, diphthericin type and, on the other hand, bacteriophage type, serotype and biochemical activity. PMID:4203597

  1. Effect of a single tetanus-diphtheria vaccine dose on the immunity of elderly people in São Paulo, Brazil.

    PubMed

    Weckx, L Y; Divino-Goes, K; Lihama, D M; Carraro, E; Bellei, N; Granato, C F H; Moraes-Pinto, M I de

    2006-04-01

    Epidemiological data regarding tetanus and diphtheria immunity in elderly people in Brazil are scarce. During the First National Immunization Campaign for the Elderly in Brazil in April 1999, 98 individuals (median age: 84 years) received one tetanus-dyphtheria (Td) vaccine dose (Butantan Institute, lot number 9808079/G). Inclusion criteria were elderly individuals without a history of severe immunosuppressive disease, acute infectious disease or use of immunomodulators. Blood samples were collected immediately before the vaccine and 30 days later. Serum was separated and stored at -20 degrees C until analysis. Tetanus and diphtheria antibodies were measured by the double-antigen ELISA test. Tetanus and diphtheria antibody concentrations lower than 0.01 IU/mL were considered to indicate the absence of protection, between 0.01 and 0.09 IU/mL were considered to indicate basic immunity, and values of 0.1 IU/mL or higher were considered to indicate full protection. Before vaccination, 18% of the individuals were susceptible to diphtheria and 94% were susceptible to tetanus. After one Td dose, 78% became fully immune to diphtheria, 13% attained basic immunity, and 9% were still susceptible to the disease. In contrast, 79% remained susceptible to tetanus, 4% had basic immunity and 17% were fully immune. Although one Td dose increases immunity to diphtheria in many elderly people who live in Brazil, a complete vaccination series appears to be necessary for the prevention of tetanus.

  2. External quality assessment for the determination of diphtheria antitoxin in human serum.

    PubMed

    Di Giovine, Paolo; Pinto, Antonella; Olander, Rose-Marie; Sesardic, Dorothea; Stickings, Paul; Berbers, Guy; Neal, Shona; Efstratiou, Androulla; Paberza, Ruta; Dauksiene, Snieguole; Bujko, Marina; Detcheva, Antoaneta; Joks, Unna; Levent, Belkis; von Hunolstein, Christina

    2010-08-01

    Accurate determination of diphtheria toxin antibodies is of value in determining the rates of immunity within broad populations or the immune status of individuals who may be at risk of infection, by assessing responses to vaccination and immunization schedule efficacy. Here we report the results of an external quality assessment (EQA) study for diphtheria serology, performed within the dedicated surveillance network DIPNET. Twelve national laboratories from 11 European countries participated by testing a standard panel of 150 sera using their current routine method: Vero cell neutralization test (NT), double-antigen enzyme-linked immunosorbent assay (ELISA; DAE), dual double-antigen time-resolved fluorescence immunoassay (dDA-DELFIA), passive hemagglutination assay (PHA), toxin binding inhibition assay (ToBI), and in-house or commercial ELISAs. The objective of the study was not to identify the best assay, as the advantages and drawbacks of methods used were known, but to verify if laboratories using their routine method would have categorized (as negative, equivocal, or positive) a serum sample in the same way. The performance of each laboratory was determined by comparing its results on a quantitative and qualitative basis to NT results from a single reference laboratory, as this test is considered the in vitro "gold standard." The performance of laboratories using NT was generally very good, while the laboratories' performance using other in vitro methods was variable. Laboratories using ELISA and PHA performed less well than those using DAE, dDA-DELFIA, or ToBI. EQA is important for both laboratories that use in vitro nonstandardized methods and those that use commercial ELISA kits.

  3. SubMICs of penicillin and erythromycin enhance biofilm formation and hydrophobicity of Corynebacterium diphtheriae strains.

    PubMed

    Gomes, D L R; Peixoto, R S; Barbosa, E A B; Napoleão, F; Sabbadini, P S; dos Santos, K R N; Mattos-Guaraldi, A L; Hirata, R

    2013-05-01

    Subinhibitory concentrations (subMICs) of antibiotics may alter bacterial surface properties and change microbial physiology. This study aimed to investigate the effect of a subMIC (⅛ MIC) of penicillin (PEN) and erythromycin (ERY) on bacterial morphology, haemagglutinating activity, cell-surface hydrophobicity (CSH) and biofilm formation on glass and polystyrene surfaces, as well as the distribution of cell-surface acidic anionic residues of Corynebacterium diphtheriae strains (HC01 tox(-) strain; CDC-E8392 and 241 tox(+) strains). All micro-organisms tested were susceptible to PEN and ERY. Growth in the presence of PEN induced bacterial filamentation, whereas subMIC of ERY caused cell-size reduction of strains 241 and CDC-E8392. Adherence to human erythrocytes was reduced after growth in the presence of ERY, while CSH was increased by a subMIC of both antibiotics in bacterial adherence to n-hexadecane assays. Conversely, antibiotic inhibition of biofilm formation was not observed. All strains enhanced biofilm formation on glass after treatment with ERY, while only strain 241 increased glass adherence after cultivation in the presence of PEN. Biofilm production on polystyrene surfaces was improved by ⅛ MIC of ERY. After growth in the presence of both antimicrobial agents, strains 241 and CDC-E8392 exhibited anionic surface charges with focal distribution. In conclusion, subMICs of PEN and ERY modified bacterial surface properties and enhanced not only biofilm formation but also cell-surface hydrophobicity. Antibiotic-induced biofilm formation may contribute to the inconsistent success of antimicrobial therapy for C. diphtheriae infections.

  4. The sero-epidemiology of diphtheria in Western Europe. ESEN Project. European Sero-Epidemiology Network.

    PubMed

    Edmunds, W J; Pebody, R G; Aggerback, H; Baron, S; Berbers, G; Conyn-van Spaendonck, M A; Hallander, H O; Olander, R; Maple, P A; Melker, H E; Olin, P; Fievret-Groyne, F; Rota, C; Salmaso, S; Tischer, A; von-Hunolstein, C; Miller, E

    2000-08-01

    Seven countries in Western Europe collected large, representative serum banks across the entire age range and tested them for diphtheria anti-toxin (sample size ranged from 2991 to 7715). Although a variety of assays were used, the results were all standardized to those of a reference laboratory and expressed in international units. The standardization process, and the availability of similar, large data sets allowed comparative analyses to be performed in which a high degree of confidence could be ascribed to observed epidemiological differences. The results showed that there were large differences in the proportion of adults with insufficient levels of protection amongst different countries. For instance, roughly 35% of 50- to 60-year-olds were found to be seronegative (titre < or = 0.01 IU/ml) in Finland compared with 70-75% in the United Kingdom. Furthermore, the proportion of seronegative adults would be expected to increase in some countries, notably Italy and the western part of Germany. In those countries with vaccination of military recruits there was a marked sex-related difference in the proportion of seropositive individuals. All countries have high levels of infant vaccine coverage (> 90%) but the accelerated schedule in the United Kingdom appears to result in lower anti-toxin titres than elsewhere. In Sweden, booster doses are not offered until 10 years of age which results in large numbers of children with inadequate levels of protection. Although the United Kingdom and Sweden both have higher proportions of seronegative children than elsewhere the likelihood of a resurgence of diphtheria in these countries seems remote.

  5. Evaluation of toxin neutralisation in test systems for diphtheria antibody assessment.

    PubMed

    Vandenberg, J; van der Gun, J W; Hendriksen, C F

    1999-01-01

    Over the past years, various authors have reported that the amount of toxin used in toxin neutralisation (TN) assays for diphtheria appears to influence the resulting relative antibody titre. Antibody affinity is thought to be an influencing factor. To confirm this observation and study the underlying mechanism of toxin neutralisation, a panel of sera was generated, differing in species of origin (mouse, guinea pig, and rabbit) and in affinity by using different immunisation schedules. The panel was then tested in relevant TN test systems for diphtheria antibody titration, namely the VERO cell test, the Toxin Binding Inhibition (ToBI) assay and the in vivo skin test in guinea pigs. A hyperimmune equine reference serum was used as the standard. Antibody affinity was measured in two different affinity ELISAs, the ammonium thiocyanate elution ELISA and the diethylamine inhibition ELISA. The VERO cell test clearly demonstrates the phenomenon; the higher the toxin dose used in the assay, the higher the resulting relative potency. The difference in relative antibody titre decreases as antibody affinity increases. This is especially evident when an equine hyperimmune reference serum is used as the standard. When a species homologous reference is used, the phenomenon is less distinct. The ToBI test, however, does not show the phenomenon. This discrepancy between these two test systems is being further investigated, and comparison will be made with the in vivo TN test. The findings confirm and support earlier observations. It is still unclear exactly which mechanisms are involved in the toxin neutralisation process. Antibody subclasses and class switching could play a role and will be further studied.

  6. The sero-epidemiology of diphtheria in Western Europe. ESEN Project. European Sero-Epidemiology Network.

    PubMed Central

    Edmunds, W. J.; Pebody, R. G.; Aggerback, H.; Baron, S.; Berbers, G.; Conyn-van Spaendonck, M. A.; Hallander, H. O.; Olander, R.; Maple, P. A.; Melker, H. E.; Olin, P.; Fievret-Groyne, F.; Rota, C.; Salmaso, S.; Tischer, A.; von-Hunolstein, C.; Miller, E.

    2000-01-01

    Seven countries in Western Europe collected large, representative serum banks across the entire age range and tested them for diphtheria anti-toxin (sample size ranged from 2991 to 7715). Although a variety of assays were used, the results were all standardized to those of a reference laboratory and expressed in international units. The standardization process, and the availability of similar, large data sets allowed comparative analyses to be performed in which a high degree of confidence could be ascribed to observed epidemiological differences. The results showed that there were large differences in the proportion of adults with insufficient levels of protection amongst different countries. For instance, roughly 35% of 50- to 60-year-olds were found to be seronegative (titre < or = 0.01 IU/ml) in Finland compared with 70-75% in the United Kingdom. Furthermore, the proportion of seronegative adults would be expected to increase in some countries, notably Italy and the western part of Germany. In those countries with vaccination of military recruits there was a marked sex-related difference in the proportion of seropositive individuals. All countries have high levels of infant vaccine coverage (> 90%) but the accelerated schedule in the United Kingdom appears to result in lower anti-toxin titres than elsewhere. In Sweden, booster doses are not offered until 10 years of age which results in large numbers of children with inadequate levels of protection. Although the United Kingdom and Sweden both have higher proportions of seronegative children than elsewhere the likelihood of a resurgence of diphtheria in these countries seems remote. PMID:11057967

  7. Diphtheria toxin resistance in human lymphocytes and lymphoblasts in the in vivo somatic cell mutation test

    SciTech Connect

    Tomkins, D.J.; Wei, L.; Laurie, K.E.

    1985-01-01

    It has been shown that circulating peripheral blood lymphocytes can be used for the enumeration of 6-thioguanine-resistant cells that presumably arise by mutation in vivo. This somatic cell mutation test has been studied in lymphocytes from human populations exposed to known mutagens and/or carcinogens. The sensitivity of the test could be further enhanced by including other gene markers, since there is evidence for locus-specific differences in response to mutagens. Resistance to diphtheria toxin (Dip/sup r/) seemed like a potential marker to incorporate into the test because the mutation acts codominantly, can readily be selected in human diploid fibroblasts and Chinese hamster cells with no evidence for cell density or cross-feeding effects, and can be assayed for in nondividing cells by measuring protein synthesis inhibition. Blood samples were collected from seven individuals, and fresh, cryopreserved, or Epstein-Barr virus (EBV)-transformed lymphocytes were tested for continued DNA synthesis (TH-thymidine, autoradiography) or protein synthesis (TVS-methionine, scintillation counting). Both fresh and cryopreserved lymphocytes, stimulated to divide with phytohemagglutinin (PHA), continued to synthesize DNA in the presence of high doses of diphtheria toxin (DT). Similarly, both dividing (PHA-stimulated) and nondividing fresh lymphocytes carried on significant levels of protein synthesis even 68 hr after exposure to 100 flocculating units (LF)/ml DT. The results suggest that human T and B lymphocytes may not be as sensitive to DT protein synthesis inhibition as human fibroblast and Chinese hamster cells. For this reason, Dip/sup r/ may not be a suitable marker for the somatic cell mutation test.

  8. Diphtheria Symptoms

    MedlinePlus

    ... into and attach to the lining of the respiratory system, which includes parts of the body that help ... neck The poison destroys healthy tissues in the respiratory system. Within two to three days, the dead tissue ...

  9. Effect of Spirulina (Arthrospira) supplementation on the immune response to tetanus toxoid vaccination in a mouse model.

    PubMed

    Chu, Wan-Loy; Quynh, Le Van; Radhakrishnan, Ammu Kutty

    2013-09-01

    The aim of this study was to investigate whether Spirulina (Arthrospira) supplementation could enhance the immune response to tetanus toxoid (TT) vaccine in a mouse model. Vaccination of TT was performed on day 7 and 21 in mice fed daily with Spirulina (50 and 150 mg/kg body weight). Both Spirulina supplementation and TT vaccination did not significantly affect body weight gain of the mice. Supplementation of Spirulina significantly enhanced IgG level (p = .01) after the first but not after the second TT vaccination. The anti-TT IgG levels of the groups that received low dose and high dose of Spirulina were not significantly different. Spirulina supplementation did not show significant effects on in vitro splenocyte proliferation and cytokine (IFN-γ and IL-4) production induced by Con A and TT. This study showed that Spirulina supplementation could enhance primary immune response in terms of antibody production, but not secondary immune response following TT vaccination in a mouse model.

  10. Impairment of the humoral and CD4(+) T cell responses in HTLV-1-infected individuals immunized with tetanus toxoid.

    PubMed

    Souza, Anselmo; Santos, Silvane; Carvalho, Lucas P; Grassi, Maria Fernanda R; Carvalho, Edgar M

    2016-08-01

    T cells from HTLV-1-infected individuals have a decreased ability to proliferate after stimulation with recall antigens. This abnormality may be due to the production of regulatory cytokine or a dysfunctional antigen presentation. The aims of this study were to evaluate the antibody production and cytokine expression by lymphocytes before and after immunization with tetanus toxoid (TT) and to evaluate the immune response of monocytes after stimulation with TT and frequency of dendritic cells (DC) subsets. HTLV-1 carriers (HC) and uninfected controls (UC) with negative serology for TT were immunized with TT, and the antibody titers were determined by ELISA as well as the cell activation markers expression by monocytes. The frequencies of DC subsets were determined by flow cytometry. Following immunization, the IgG anti-TT titers and the frequency of CD4(+) T cells expressing IFN-γ, TNF-α and IL-10 in response to TT were lower in the HC than in the UC. Additionally, monocytes from HC did not exhibit increased HLA-DR expression after stimulation with TT, and presented low numbers of DC subsets, therefore, it's necessary to perform functional studies with antigen-presenting cells. Collectively, our finding suggests that HC present an impairment of the humoral and CD4(+) T cell immune responses after vaccination.

  11. Tetanus toxoid-loaded layer-by-layer nanoassemblies for efficient systemic, mucosal, and cellular immunostimulatory response following oral administration.

    PubMed

    Harde, Harshad; Agrawal, Ashish Kumar; Jain, Sanyog

    2015-10-01

    The present study reports the tetanus toxoid (TT)-loaded layer-by-layer nanoassemblies (layersomes) with enhanced protection, permeation, and presentation for comprehensive oral immunization. The stable and lyophilized TT-loaded layersomes were prepared by a thin-film hydration method followed by alternate layer-by-layer coating of an electrolyte. The developed system was assessed for in vitro stability of antigen and formulation, cellular uptake, ex vivo intestinal uptake, and immunostimulatory response using a suitable experimental protocol. Layersomes improved the stability in simulated biological media as well as protected the integrity/conformation and native 3D structure of TT as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorescence spectroscopy, respectively. The cell culture studies demonstrated a 3.8-fold higher permeation of layersomes in Caco-2 cells and an 8.5-fold higher uptake by antigen-presenting cells (RAW 264.7). The TT-loaded layersomes elicited a complete immunostimulatory profile consisting of higher systemic (serum IgG titer), mucosal (sIgA titer), and cellular (interleukin-2 (IL-2) and interferon-γ (IFN-γ) levels) immune response after peroral administration in mice. The modified TT inhibition assay further confirmed the elicitation of complete protective levels of anti-TT antibody (>0.1 IU/mL) by layersomes. In conclusion, the proposed strategy is expected to contribute significantly in the field of stable liposome technology for mass immunization through the oral route.

  12. Haemophilus influenzae type b-Neisseria meningitidis serogroups C and Y tetanus toxoid conjugate vaccine for infants and toddlers.

    PubMed

    Bryant, Kristina A; Marshall, Gary S

    2011-07-01

    The highest rates of invasive meningococcal disease occur in children under 2 years of age, yet as of early 2011 no vaccine was licensed for the youngest infants. However, a novel vaccine consisting of capsular polysaccharides from Haemophilus influenzae type b (Hib) and Neisseria meningitidis serogroups C and Y conjugated to tetanus toxoid (HibMenCY-TT; MenHibrix, GlaxoSmithKline) is in the late stages of development. In clinical trials involving more than 7800 children, HibMenCY-TT was shown to be safe and immunogenic when administered at 2, 4, 6 and 12-15 months of age. Anti-polyribosylribitol phosphate antibody responses were noninferior to those elicited by licensed monovalent Hib vaccines, and most vaccinees developed bactericidal antibodies against N. meningitidis serogroups C and Y. The majority of subjects retained antibody responses as far as 3 years after vaccination. If licensed, HibMenCY-TT not only represents an incremental option for protection against invasive Hib, but also has the potential to prevent invasive meningococcal disease without increasing the number of injections.

  13. Similarity of rpoB gene sequences of sucrose-fermenting and non-fermenting Corynebacterium diphtheriae strains.

    PubMed

    Hirata, R; Pacheco, L G; Soares, S C; Santos, L S; Moreira, L O; Sabbadini, P S; Santos, C S; Miyoshi, A; Azevedo, V A; Mattos-Guaraldi, A L

    2011-03-01

    During the last decades, the majority of Brazilian Corynebacterium diphtheriae isolates were shown to be capable to metabolize sucrose, sometimes leading to erroneous identification as a non-diphtheric Corynebacterium species. The sequencing of the polymorphic region of the RNA polymerase beta subunit-encoding gene (rpoB) is an important taxonomic tool for identification of corynebacteria. The present study aimed to investigate the rpoB gene polymorphic features of sucrose-fermenting and non sucrose-fermenting strains. The results showed that sucrose-fermenting strains presented rpoB gene polymorphic regions with more than 98% similarity with the sequences deposited in the gene bank corresponding to non sucrose-fermenting strains. Data indicate that sucrose-fermenting isolates may act as a variant of C. diphtheriae biotype mitis. In addition we alert that sucrose-fermenting strains should not be discarded as contaminants mainly in countries where the possibility of isolation of this variant is higher.

  14. Pilus gene pool variation and the virulence of Corynebacterium diphtheriae clinical isolates during infection of a nematode.

    PubMed

    Broadway, Melissa M; Rogers, Elizabeth A; Chang, Chungyu; Huang, I-Hsiu; Dwivedi, Prabhat; Yildirim, Suleyman; Schmitt, Michael P; Das, Asis; Ton-That, Hung

    2013-08-01

    Toxigenic Corynebacterium diphtheriae strains cause diphtheria in humans. The toxigenic C. diphtheriae isolate NCTC13129 produces three distinct heterotrimeric pili that contain SpaA, SpaD, and SpaH, making up the shaft structure. The SpaA pili are known to mediate bacterial adherence to pharyngeal epithelial cells. However, to date little is known about the expression of different pili in various clinical isolates and their importance in bacterial pathogenesis. Here, we characterized a large collection of C. diphtheriae clinical isolates for their pilin gene pool by PCR and for the expression of the respective pilins by immunoblotting with antibodies against Spa pilins. Consistent with the role of a virulence factor, the SpaA-type pili were found to be prevalent among the isolates, and most significantly, corynebacterial adherence to pharyngeal epithelial cells was strictly correlated with isolates that were positive for the SpaA pili. By comparison, the isolates were heterogeneous for the presence of SpaD- and SpaH-type pili. Importantly, using Caenorhabditis elegans as a model host for infection, we show here that strain NCTC13129 rapidly killed the nematodes, the phenotype similar to isolates that were positive for toxin and all pilus types. In contrast, isogenic mutants of NCTC13129 lacking SpaA-type pili or devoid of toxin and SpaA pili exhibited delayed killing of nematodes with similar kinetics. Consistently, nontoxigenic or toxigenic isolates that lack one, two, or all three pilus types were also attenuated in virulence. This work signifies the important role of pili in corynebacterial pathogenesis and provides a simple host model to identify additional virulence factors.

  15. Immunogenicity of one, two or three doses of a meningococcal C conjugate vaccine conjugated to tetanus toxoid, given as a three-dose primary vaccination course in UK infants at 2, 3 and 4 months of age with acellular pertussis-containing DTP/Hib vaccine.

    PubMed

    Southern, J; Crowley-Luke, A; Borrow, R; Andrews, N; Miller, E

    2006-01-12

    Reduction of the number of injections necessary to confer protection in the infant schedule would reduce discomfort, improve cost-effectiveness and create space for the addition of new vaccinations in the future. This study assessed the immunogenicity of one, two or three doses of meningococcal C conjugate vaccine conjugated to tetanus toxoid (MCC-TT) [Neis-VacC] given concomitantly with a combined diphtheria/tetanus/acellular pertussis/Haemophilus influenzae type b -TT conjugate (DTaP-Hib-TT) [Infanrix-Hib] vaccine at 2, 3 and 4 months of age. A total of 106 healthy UK infants were enrolled and randomised into two groups, one in which blood was taken after the first and third dose and the other after the second and third dose. The meningococcal serogroup C serum bactericidal antibody (SBA) geometric mean titre (GMT) rose significantly from post-first dose (491, 95% CI 275, 877) to post-second dose (1052, 95% CI 774, 1433) (p=0.03), with no significant change after the third dose (1024, 95% CI 768, 1366). An SBA titre of >or=8 was achieved by 92% after the first dose and 100% after the second and third doses. The Hib IgG geometric mean concentration (GMC) rose significantly after each dose: post-first (0.14 microg/ml 95% CI 0.10, 0.18), post-second (0.54 microg/ml, 95% CI 0.33, 0.90), post-third (2.04 microg/ml, 95% CI 1.52, 2.74). The Hib GMC after the third dose was higher than reported previously when this DTaP/Hib was given either on its own or concomitantly with a MCC-CRM conjugate vaccine according to the UK 2, 3 and 4 month schedule. This suggests some enhancement of the response to a Hib-TT vaccine by concomitant administration of MCC-TT. These results suggest that a reduced number of doses of MCC-TT would be adequate in infancy if given concomitantly with an acellular pertussis-containing vaccine.

  16. Determination of the minimal essential nucleotide sequence for diphtheria tox repressor binding by in vitro affinity selection.

    PubMed

    Tao, X; Murphy, J R

    1994-09-27

    The expression of diphtheria toxin in lysogenic toxigenic strains of Corynebacterium diphtheriae is controlled by the heavy metal ion-activated regulatory protein DtxR. In the presence of divalent heavy metal ions, DtxR specifically binds to the diphtheria tox operator and protects a 27-bp interrupted palindromic sequence from DNase I digestion. To determine the consensus DNA sequence for DtxR binding, we have used gel electrophoresis mobility-shift assay and polymerase chain reaction (PCR) amplification for in vitro affinity selection of DNA binding sequences from a universe of 6.9 x 10(10) variants. After 10 rounds of in vitro affinity selection, each round coupled with 30 cycles of PCR amplification, we isolated and characterized a family of DNA sequences that function as DtxR-responsive genetic elements both in vitro and in vivo. Moreover, these DNA sequences were found to bind activated DtxR with an affinity similar to that of the wild-type tox operator. The DNA sequence analysis of 21 unique in vitro affinity-selected binding sites has revealed the minimal essential nucleotide sequence for DtxR binding to be a 9-bp palindrome separated by a single base pair.

  17. Genotypic and phenotypic characteristics of Corynebacterium diphtheriae strains isolated from patients in belarus during an epidemic period.

    PubMed

    Titov, Leonid; Kolodkina, Valentina; Dronina, Alina; Grimont, Francine; Grimont, Patrick A D; Lejay-Collin, Monique; de Zoysa, Aruni; Andronescu, Constantin; Diaconescu, Angela; Marin, Byanca; Efstratiou, Androulla

    2003-03-01

    One hundred two Corynebacterium diphtheriae strains (93 of the gravis biotype and nine of the mitis biotype) isolated from clinical cases during the Belarus diphtheria epidemic were characterized by biotyping, toxigenicity testing by the Elek test and an indirect hemagglutination assay, phage typing, and ribotyping. The gravis biotype strains were characterized as high and medium toxin producers, and strains of biotype mitis were characterized as low and medium toxin producers. Most strains (82 of 102) were distributed among five phage types. Seventy-two strains (64 of the gravis biotype and 8 of the mitis biotype) belonged to phage type VI ls5,34add. Hybridization of genomic DNA digested with BstEII and PvuII revealed five ribotype patterns, namely, D1, D4, D6, D7, and D13. The majority of gravis biotype strains belonged to ribotypes D1 (49 of 93) and D4 (33 of 93) and included one clonal group of C. diphtheriae. This clone predominated in all regions in Belarus. There was a statistical association between ribotypes and phage types but not between ribotypes and levels of toxin production.

  18. pH-dependent conformational changes of diphtheria toxin adsorbed to lipid monolayers by neutron and X-ray reflection

    NASA Astrophysics Data System (ADS)

    Kent, Michael; Yim, Hyun; Satija, Sushil; Kuzmenko, Ivan

    2006-03-01

    Several important bacterial toxins, such as diphtheria, tetanus, and botulinum, invade cells through a process of high affinity binding, internalization via endosome formation, and subsequent membrane penetration of the catalytic domain activated by a pH drop in the endosome. These toxins are composed of three domains: a binding domain, a translocation domain, and an enzyme. The translocation process is not well understood with regard to the detailed conformational changes that occur at each step, To address this, we performed neutron reflectivity measurements for diphtheria toxin bound to lipid monolayers as a function of pH. While the final membrane inserted conformation will not be reproduced with the present monolayer system, important insights can still be gained into several intermediate stages. In particular, we show that no adsorption occurs at pH = 7.6, but strong adsorption occurs over at a pH range from 6.5 to 6.0. Following binding, at least two stages of conformational change occur, as the thickness increases from pH 6.3 to 5.3 and then decreases from pH 5.3 to 4.5. In addition, the dimension of the adsorbed layer substantially exceeds that of the largest dimension in the crystal structure of monomeric diphtheria, suggesting that the toxin may be present as multimers.

  19. Diphtheria Toxin-Induced Cell Death Triggers Wnt-Dependent Hair Cell Regeneration in Neonatal Mice

    PubMed Central

    Hu, Lingxiang; Lu, Jingrong; Chiang, Hao; Shi, Fuxin

    2016-01-01

    Cochlear hair cells (HCs), the sensory cells that respond to sound, do not regenerate after damage in adult mammals, and their loss is a major cause of deafness. Here we show that HC regeneration in newborn mouse ears occurred spontaneously when the original cells were ablated by treatment with diphtheria toxin (DT) in ears that had been engineered to overexpress the DT receptor, but was not detectable when HCs were ablated in vivo by the aminoglycoside antibiotic neomycin. A variety of Wnts (Wnt1, Wnt2, Wnt2b, Wnt4, Wnt5a, Wnt7b, Wnt9a, Wnt9b, and Wnt11) and Wnt pathway component Krm2 were upregulated after DT damage. Nuclear β-catenin was upregulated in HCs and supporting cells of the DT-damaged cochlea. Pharmacological inhibition of Wnt decreased spontaneous regeneration, confirming a role of Wnt signaling in HC regeneration. Inhibition of Notch signaling further potentiated supporting cell proliferation and HC differentiation that occurred spontaneously. The absence of new HCs in the neomycin ears was correlated to less robust Wnt pathway activation, but the ears subjected to neomycin treatment nonetheless showed increased cell division and HC differentiation after subsequent forced upregulation of β-catenin. These studies suggest, first, that Wnt signaling plays a key role in regeneration, and, second, that the outcome of a regenerative response to damage in the newborn cochlea is determined by reaching a threshold level of Wnt signaling rather than its complete absence or presence. SIGNIFICANCE STATEMENT Sensory HCs of the inner ear do not regenerate in the adult, and their loss is a major cause of deafness. We found that HCs regenerated spontaneously in the newborn mouse after diphtheria toxin (DT)-induced, but not neomycin-induced, HC death. Regeneration depended on activation of Wnt signaling, and regeneration in DT-treated ears correlated to a higher level of Wnt activation than occurred in nonregenerating neomycin-treated ears. This is significant

  20. Enhanced immunogenicity of a tricomponent mannan tetanus toxoid conjugate vaccine targeted to dendritic cells via Dectin-1 by incorporating β-glucan.

    PubMed

    Lipinski, Tomasz; Fitieh, Amira; St Pierre, Joëlle; Ostergaard, Hanne L; Bundle, David R; Touret, Nicolas

    2013-04-15

    In a previous attempt to generate a protective vaccine against Candida albicans, a β-mannan tetanus toxoid conjugate showed poor immunogenicity in mice. To improve the specific activation toward the fungal pathogen, we aimed to target Dectin-1, a pattern-recognition receptor expressed on monocytes, macrophages, and dendritic cells. Laminarin, a β-glucan ligand of Dectin-1, was incorporated into the original β-mannan tetanus toxoid conjugate providing a tricomponent conjugate vaccine. A macrophage cell line expressing Dectin-1 was employed to show binding and activation of Dectin-1 signal transduction pathway by the β-glucan-containing vaccine. Ligand binding to Dectin-1 resulted in the following: 1) activation of Src family kinases and Syk revealed by their recruitment and phosphorylation in the vicinity of bound conjugate and 2) translocation of NF-κB to the nucleus. Treatment of immature bone marrow-derived dendritic cells (BMDCs) with tricomponent or control vaccine confirmed that the β-glucan-containing vaccine exerted its enhanced activity by virtue of dendritic cell targeting and uptake. Immature primary cells stimulated by the tricomponent vaccine, but not the β-mannan tetanus toxoid vaccine, showed activation of BMDCs. Moreover, treated BMDCs secreted increased levels of several cytokines, including TGF-β and IL-6, which are known activators of Th17 cells. Immunization of mice with the novel type of vaccine resulted in improved immune response manifested by high titers of Ab recognizing C. albicans β-mannan Ag. Vaccine containing laminarin also affected distribution of IgG subclasses, showing that vaccine targeting to Dectin-1 receptor can benefit from augmentation and immunomodulation of the immune response.

  1. Immunogenicity of HIV-1 IIIB and SHIV 89.6P Tat and Tat toxoids in rhesus macaques: induction of humoral and cellular immune responses.

    PubMed

    Richardson, Max W; Mirchandani, Jyotika; Silvera, Peter; Régulier, Emmanuel G; Capini, Christelle; Bojczuk, Paul M; Hu, Jason; Gracely, Edward J; Boyer, Jean D; Khalili, Kamel; Zagury, Jean-François; Lewis, Mark G; Rappaport, Jay

    2002-09-01

    This study compared immune responses in rhesus macaques immunized with unmodified HIV-1 IIIB Tat, SHIV89.6P Tat, and carboxymethylated IIIB and 89.6P Tat toxoids. Immunization with either IIIB or 89.6P preparation induced high titer and broadly crossreactive serum anti-Tat IgG that recognized HIV-1 subtype-E and SIVmac251 Tat. However, the response was delayed, and titers were lower in 89.6P vaccination groups. Serum anti-Tat IgG recognized peptides corresponding to the amino-terminus, basic domain, and carboxy-terminal region. Cellular proliferative responses to Tat toxoids corresponding to the immunogen were evident in vitro in both IIIB and 89.6P groups. Crossreactive proliferative responses were observed in IIIB groups in response to stimulation with 89.6P or SIVmac251 Tat toxoids, but were much less prevalent in 89.6P groups. The truncated 86 amino acid IIIB Tat appears to be more immunogenic than the 102 amino acid 89.6P Tat with respect to both humoral and cellular immune responses, and may be a better vaccine component. Despite induction of robust humoral and cellular immune responses (including both CD4+ and CD8+ T-cell responses) to Tat, all animals were infected upon intravenous challenge with 30 MID(50) of SHIV89.6P and outcome of vaccine groups was not different from controls. Sequencing both Tat exons from serum viral RNA revealed no evidence of escape mutants. These results suggest that with intravenous SHIV89.6P challenge in rhesus macaques, precipitous CD4+ T-cell decline overwhelms potentially protective immune responses. Alternatively, Tat specific CD8+ T-cell responses may not appropriately recognize infected cells in vivo in this model. In view of evidence demonstrating Tat specific CTLs in the SIV model and in humans infected with HIV-1, results in this pathogenic SHIV model may not apparently predict the efficacy of this approach in human studies. The potency and cross-reactivity of these immune responses confirm Tat toxoid as an excellent

  2. Development of a recombinant epsilon toxoid vaccine against enterotoxemia and its use as a combination vaccine with live attenuated sheep pox virus against enterotoxemia and sheep pox.

    PubMed

    Chandran, Dev; Naidu, Sureddi Satyam; Sugumar, Parthasarathy; Rani, Gudavalli Sudha; Vijayan, Shahana Pallichera; Mathur, Deepika; Garg, Lalit C; Srinivasan, Villuppanoor Alwar

    2010-06-01

    Sheep pox and enterotoxemia are important diseases of sheep, and these diseases cause severe economic losses to sheep farmers. The present study was undertaken to evaluate the potential of formaldehyde-inactivated recombinant epsilon toxin as a vaccine candidate. The potency of the recombinant epsilon toxoid with aluminum hydroxide as an adjuvant in sheep was determined. Vaccinated sheep were protected against enterotoxemia, with potency values of >5 IU being protective. Further, the use of this construct in a combination vaccine against sheep pox resulted in the sheep being protected against both sheep pox and enterotoxemia.

  3. Nitrogen-gas bubbling during the cultivation of Clostridium tetani produces a higher yield of tetanus toxin for the preparation of its toxoid.

    PubMed

    De Luca, M M; Abeiro, H D; Bernagozzi, J A; Basualdo, J A

    1997-01-01

    We investigated the effect of exposing cultures of Clostridium tetani to nitrogen (N2) gas on the recovery of tetanus toxin to be processed for the preparation of its toxoid. N2 was bubbled through nine 10-liter cultures during the growth of the bacteria, while nine parallel control incubations were maintained without bubbling. We found that treatment of the C. tetani anaerobes with an inert gas in this manner during cultivation produced a highly significant increase in the yield of tetanus toxin from them in comparison with the standard procedure.

  4. Men with low vitamin A stores respond adequately to primary yellow fever and secondary tetanus toxoid vaccination.

    PubMed

    Ahmad, Shaikh M; Haskell, Marjorie J; Raqib, Rubhana; Stephensen, Charles B

    2008-11-01

    Current recommendations for vitamin A intake and liver stores (0.07 micromol/g) are based on maintaining normal vision. Higher levels may be required for maintaining normal immune function. The objective of this study was to assess the relationship between total body vitamin A stores in adult men and measures of adaptive immune function. We conducted an 8-wk residential study among 36 healthy Bangladeshi men with low vitamin A stores. Subjects received a standard diet and were randomized in a double-blind fashion to receive vitamin A (240 mg) or placebo during wk 2 and 3. Subjects received Yellow Fever Virus (YFV) and tetanus toxoid (TT) vaccines during wk 5. Vitamin A stores were estimated by isotopic dilution during wk 8. Vaccine-specific lymphocyte proliferation, cytokine production, and serum antibody responses were evaluated before and after vaccination. Vitamin A supplementation increased YFV- and TT-specific lymphocyte proliferation and YFV-specific interleukin (IL)-5, IL-10, and tumor necrosis factor-alpha production but inhibited development of a TT-specific IL-10 response. Both groups developed protective antibody responses to both vaccines. Some responses correlated positively with vitamin A stores. These findings indicate that the currently recommended vitamin A intake is sufficient to sustain a protective response to YFV and TT vaccination. However, YFV-specific lymphocyte proliferation, some cytokine responses, and neutralizing antibody were positively associated with liver vitamin A stores > 0.084 micromol/g. Such increases may enhance vaccine protection but raise the question of whether immune-mediated chronic diseases may by exacerbated by high-level dietary vitamin A.

  5. Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans.

    PubMed

    Santos, Louisy Sanches Dos; Antunes, Camila Azevedo; Santos, Cintia Silva Dos; Pereira, José Augusto Adler; Sabbadini, Priscila Soares; Luna, Maria das Graças de; Azevedo, Vasco; Hirata Júnior, Raphael; Burkovski, Andreas; Asad, Lídia Maria Buarque de Oliveira; Mattos-Guaraldi, Ana Luíza

    2015-08-01

    Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO32-) is toxic to most microorganisms, TeO32--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO32--resistance (TeR) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative TeR determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO32- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegans and the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the TeR and pathogenic potential of C. diphtheriae.

  6. THE RELATION OF THE TOXICITY OF DIPHTHERIA TOXIN TO ITS NEUTRALIZING VALUE UPON ANTITOXIN AT DIFFERENT STAGES IN THE GROWTH OF CULTURE.

    PubMed

    Park, W H; Atkinson, J P

    1898-07-01

    Until recently diphtheria " toxin " was supposed to be a single definite substance and to have a definite toxicity in animals and neutralizing power for antitoxin. A fatal dose of toxin, without regard to the conditions under which it was produced or preserved, was supposed to require always the same quantity of antitoxin to neutralize it. Ehrlich's researches have completely done away with this theory, and have substituted for it one which assumes the toxin to be only at its origin a single definite chemical compound with definite physiological and antitoxic properties. According to Ehrlich the toxin is an unstable substance which readily loses its toxicity, while at the same time its affinity for antitoxin may be either increased or decreased. Its neutralization by antitoxin he considers to be due to a chemical union between the toxin and the antitoxin. The results of our experiments as detailed in this paper are fully in accord with those published by Ehrlich, as to the varying neutralizing value of a minimal fatal dose of " toxin "; they, however, go further and indicate roughly a general law in accordance with which these changes occur. The neutralizing value of a fatal dose of toxin is at its lowest in the culture fluid when the first considerable amounts of toxin have been produced. After a short period, during which the quantity of toxin in the fluid is increasing, the neutralizing value of the fatal dose begins to increase, at first rapidly, then more slowly. While the culture is still in vigorous growth and new toxin is being produced, the neutralizing value of the fatal dose fluctuates somewhat, but with a generally upward tendency. After the cessation of toxin production the neutralizing value of the fatal dose increases steadily until it becomes five to ten times its original amount. In our experiments the greatest value for L(+) was 126, the least 27. As at 6 hours L(+) was only 72 and at 28 hours only 91, we doubt whether L(+) ever reaches above 150

  7. Opposite nucleotide usage biases in different parts of the Corynebacterium diphtheriae spaC gene.

    PubMed

    Khrustalev, Vladislav Victorovich; Barkovsky, Eugene Victorovich; Kolodkina, Valentina Leonidovna; Khrustaleva, Tatyana Aleksandrovna

    2015-01-01

    In this work we described a bacterial open reading frame with two different directions of nucleotide usage biases in its two parts. The level of GC-content in third codon positions (3GC) is equal to 40.17 ± 0.22% during the most of the length of Corynebacterium diphtheriae spaC gene. However, in the 3'-end of the same gene (from codon #1600 to codon #1873) 3GC level is equal to 64.61 ± 0.91%. Using original methodology ('VVTAK Sliding window' and 'VVTAK VarInvar') we approved that there is an ongoing mutational AT-pressure during the most of the length of spaC gene (up to codon #1599), and there is an ongoing mutational G-pressure in the 3′-end of spaC. Intragenic promoters predicted by three different methods may be the cause of the differences in preferable types of nucleotide mutations in spaC parts because of their autonomous transcription.

  8. Does tetanus-diphtheria-acellular pertussis vaccination interfere with serodiagnosis of pertussis infection?

    PubMed

    Pawloski, Lucia C; Kirkland, Kathryn B; Baughman, Andrew L; Martin, Monte D; Talbot, Elizabeth A; Messonnier, Nancy E; Tondella, Maria Lucia

    2012-06-01

    An anti-pertussis toxin (PT) IgG enzyme-linked immunosorbent assay (ELISA) was analytically validated for the diagnosis of pertussis at a cutoff of 94 ELISA units (EU)/ml. Little was known about the performance of this ELISA in the diagnosis of adults recently vaccinated with tetanus-diphtheria-acellular pertussis (Tdap) vaccine, which contains PT. The goal of this study was to determine when the assay can be used following Tdap vaccination. A cohort of 102 asymptomatic health care personnel (HCP) vaccinated with Tdap (Adacel; Sanofi Pasteur) were aged 19 to 79 years (median, 47 years) at vaccination. For each HCP, specimens were available for evaluation at 2 to 10 time points (prevaccination to 24 months postvaccination), and geometric mean concentrations (GMC) for the cohort were calculated at each time point. Among 97 HCP who responded to vaccination, a mixed-model analysis with prediction and tolerance intervals was performed to estimate the time at which serodiagnosis can be used following vaccination. The GMCs were 8, 21, and 9 EU/ml at prevaccination and 4 and 12 months postvaccination, respectively. Eight (8%) of the 102 HCP reached antibody titers of ≥94 EU/ml during their peak response, but none had these titers by 6 months postvaccination. The calculated prediction and tolerance intervals were <94 EU/ml by 45 and 75 days postvaccination, respectively. Tdap vaccination 6 months prior to testing did not confound result interpretation. This seroassay remains a valuable diagnostic tool for adult pertussis.

  9. Development of an alternative method for testing the immunogenicity of diphtheria vaccines.

    PubMed

    Manghi, M A; Pasetti, M F; Brero, M L; Deluchi, S; di Paola, G; Mathet, V; Eriksson, P V

    1995-04-01

    The immunogenicity of the diphtheria component of 73 commercial vaccines from five different manufacturers was tested by the toxin neutralization test (TNT) and the enzyme-linked immunosorbent assay (ELISA) developed in our laboratory. A comparison of the antibody levels measured by both assays showed a very good correlation (r = 0.95, p < 0.001). The results suggest that the proposed ELISA is a reliable, simple and economical alternative to the TNT in guinea pigs. Also, the ELISA was found to measure IgG antibody levels as low as 5.5 x 10(-5) IU ml-1. To evaluate the possibility of accelerating the active immunization during the activity test of vaccines, an alternative schedule using one single human dose was assayed. A very good correlation was observed between the IgG antibody response obtained with this schedule and with the traditional programme. Therefore, the cost and the time required to perform the activity test may be considerably reduced when both the rapid immunization schedule and the ELISA are used.

  10. European sero-epidemiology network: standardisation of the results of diphtheria antitoxin assays.

    PubMed

    von Hunolstein, C; Aggerbeck, H; Andrews, N; Berbers, G; Fievet-Groyne, F; Maple, P A; Olander, R M; Raux, M; Tischer, A

    2000-08-01

    A European Sero-Epidemiological Network (ESEN) was established with the aim to co-ordinate and harmonise serological surveillance of immunity to communicable diseases in Europe. In this study the inter-laboratory standardisation of diphtheria toxin antibody measurements is reported. A standard panel of 162 sera was tested by the participating laboratories using an in vitro assay of their choice: VERO cell toxin neutralisation assay (NT), double-antigen delayed time-resolved fluorescence immuno-assay (DA-DELFIA), double-antigen enzyme-linked immunosorbent assay (DAE), toxin binding inhibition test (ToBI) and an indirect enzyme-linked immunosorbent assay (ELISA). The results were standardised using regression against the NT. The variations due to inter-laboratory and inter-assay variation, which would otherwise make it difficult directly to compare the main serum bank results by the different laboratories and the various assays were successfully minimised by the standardisation. The regression equations obtained will be used to transform the respective local results of testing the main serum bank into the reference test unitages. This study also gave the opportunity to compare the various assays within and between laboratories. This demonstrated a very high correlation between DA-DELFIA, DAE, ToBI and the NT. The ELISA showed a good correlation, too, however sera below some 0.1 IU/ml were overestimated.

  11. Tetanus, diphtheria, and acellular pertussis vaccination among women of childbearing age-United States, 2013.

    PubMed

    O'Halloran, Alissa C; Lu, Peng-Jun; Williams, Walter W; Ding, Helen; Meyer, Sarah A

    2016-07-01

    The incidence of pertussis in the United States has increased since the 1990s. Tetanus, diphtheria, and acellular pertussis (Tdap) vaccination of pregnant women provides passive protection to infants. Tdap vaccination is currently recommended for pregnant women during each pregnancy, but coverage among pregnant women and women of childbearing age has been suboptimal. Data from the 2013 Behavioral Risk Factor Surveillance System (BRFSS) and 2013 National Health Interview Survey (NHIS) were used to determine national and state-specific Tdap vaccination coverage among women of childbearing age by self-reported pregnancy status at the time of the survey. Although this study could not assess coverage of Tdap vaccination received during pregnancy because questions on whether Tdap vaccination was received during pregnancy were not asked in BRFSS and NHIS, demographic and access-to-care factors associated with Tdap vaccination coverage in this population were assessed. Tdap vaccination coverage among all women 18-44 years old was 38.4% based on the BRFSS and 23.3% based on the NHIS. Overall, coverage did not differ by pregnancy status at the time of the survey. Coverage among all women 18-44 years old varied widely by state. Age, race and ethnicity, education, number of children in the household, and access-to-care characteristics were independently associated with Tdap vaccination in both surveys. We identified associations of demographic and access-to-care characteristics with Tdap vaccination that can guide strategies to improve vaccination rates in women during pregnancy.

  12. Diphtheria toxin–mediated ablation of lymphatic endothelial cells results in progressive lymphedema

    PubMed Central

    Gardenier, Jason C.; Hespe, Geoffrey E.; Kataru, Raghu P.; Savetsky, Ira L.; Torrisi, Jeremy S.; Nores, Gabriela D. García; Dayan, Joseph J.; Chang, David; Zampell, Jamie; Martínez-Corral, Inés; Ortega, Sagrario; Mehrara, Babak J.

    2016-01-01

    Development of novel treatments for lymphedema has been limited by the fact that the pathophysiology of this disease is poorly understood. It remains unknown, for example, why limb swelling resulting from surgical injury resolves initially, but recurs in some cases months or years later. Finding answers for these basic questions has been hampered by the lack of adequate animal models. In the current study, we used Cre-lox mice that expressed the human diphtheria toxin receptor (DTR) driven by a lymphatic-specific promoter in order to noninvasively ablate the lymphatic system of the hind limb. Animals treated in this manner developed lymphedema that was indistinguishable from clinical lymphedema temporally, radiographically, and histologically. Using this model and clinical biopsy specimens, we show that the initial resolution of edema after injury is dependent on the formation of collateral capillary lymphatics and that this process is regulated by M2-polarized macrophages. In addition, we show that despite these initial improvements in lymphatic function, persistent accumulation of CD4+ cells inhibits lymphangiogenesis and promotes sclerosis of collecting lymphatics, resulting in late onset of edema and fibrosis. Our findings therefore provide strong evidence that inflammatory changes after lymphatic injury play a key role in the pathophysiology of lymphedema. PMID:27699240

  13. Corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique mycothiol redox relay mechanism.

    PubMed

    Tossounian, Maria-Armineh; Pedre, Brandán; Wahni, Khadija; Erdogan, Huriye; Vertommen, Didier; Van Molle, Inge; Messens, Joris

    2015-05-01

    Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen.

  14. Successful induction of protective antibody responses against Haemophilus influenzae type b and diphtheria after transcutaneous immunization with the glycoconjugate polyribosyl ribitol phosphate-cross-reacting material 197 vaccine.

    PubMed

    Mawas, Fatme; Peyre, Marisa; Beignon, Anne-Sophie; Frost, Laura; Del Giudice, Giuseppe; Rappuoli, Rino; Muller, Sylviane; Sesardic, Dorothea; Partidos, Charalambos D

    2004-09-15

    We examined the antibody responses elicited in rats after transcutaneous immunization (TCI) with the Haemophilus influenzae type b (Hib)-cross-reacting material (CRM(197)) glycoconjugate vaccine coadministered with cholera toxin or mutants of heat-labile enterotoxin of Escherichia coli (LTK63 and LTR72) as adjuvants. The glycoconjugate vaccine was immunogenic, eliciting high antibody responses to the capsular polysaccharide of Hib and to diphtheria toxin. Passively transferred immune serum protected infant rats against challenge with the Hib Eagan strain and exhibited strong neutralizing activity against diphtheria toxin both in vitro and in vivo. The finding that TCI of rats can elicit antibody responses surpassing the minimum levels required for protective immunity against Hib and diphtheria suggests that this immunization strategy holds a lot of promise for future pediatric use. However, further studies are required to confirm the potential of TCI with glycoconjugate vaccines in humans.

  15. Active immunization of cattle with a bothropic toxoid does not abrogate envenomation by Bothrops asper venom, but increases the likelihood of survival.

    PubMed

    Herrera, María; González, Katherine; Rodríguez, Carlos; Gómez, Aarón; Segura, Álvaro; Vargas, Mariángela; Villalta, Mauren; Estrada, Ricardo; León, Guillermo

    2017-03-01

    This study assessed the protective effect of active immunization of cattle to prevent the envenomation induced by B. asper venom. Two groups of oxen were immunized with a bothropic toxoid and challenged by an intramuscular injection of either 10 or 50 mg B. asper venom, to induce moderate or severe envenomations, respectively. Non-immunized oxen were used as controls. It was found that immunized oxen developed local edema similar to those observed in non-immunized animals. However, systemic effects were totally prevented in immunized oxen challenged with 10 mg venom, and therefore antivenom treatment was not required. When immunized oxen were challenged with 50 mg venom, coagulopathy was manifested 3-16 h later than in non-immunized oxen, demonstrating a delay in the onset of systemic envenomation. In these animals, active immunization did not eliminate the need for antivenom treatment, but increased the time lapse in which antivenom administration is still effective. All experimentally envenomed oxen completely recovered after a week following venom injection. Our results suggest that immunization of cattle with a bothropic toxoid prevents the development of systemic effects in moderate envenomations by B. asper, but does not abrogate these effects in severe envenomation.

  16. Non-invasive, epicutaneous immunisation with toxoid in deformable vesicles protects mice against tetanus, chiefly owing to a Th2 response.

    PubMed

    Chopra, Amla; Cevc, Gregor

    2014-06-02

    A non-invasive, intra/transcutaneous immunisation of mice with a suitable combination of tetanus toxoid, ultradeformable vesicle (Transfersome®) carrier, and monophosphoryl lipid A adjuvant targets immuno-competent cells in a body and can protect 100% of the tested mice against an otherwise lethal (50×LD50) parenteral tetanus toxin challenge. The late immune response to the epicutaneously applied tetanus toxoid in such vesicles consists chiefly of circulating IgG1 and IgG2b antibody isotypes, indicative of a specific Th2 cellular response bias. Immunisations by subcutaneous injections moreover protect 100% of mice against a similar, otherwise lethal, dose of tetanus toxin. However, the immune response to transcutaneous and invasive immunisation differs. The latter elicits mainly IgG1 and IgG2b as well as IgG2a antibody isotypes, indicative of a mixed Th1/Th2 response. The cytokine response of the intra/transcutaneously and subcutaneously immunised mice reflects the difference in the organ-specific manner. IFN-γ concentration is appreciably increased in the draining lymph nodes and IL-10 in spleen. Since tetanus is a neutral antigen, both the Th1-specific IFN-γ and the Th-2 specific-IL-10 are observable.

  17. Utilization of host iron sources by Corynebacterium diphtheriae: multiple hemoglobin-binding proteins are essential for the use of iron from the hemoglobin-haptoglobin complex.

    PubMed

    Allen, Courtni E; Schmitt, Michael P

    2015-02-01

    The use of hemin iron by Corynebacterium diphtheriae requires the DtxR- and iron-regulated ABC hemin transporter HmuTUV and the secreted Hb-binding protein HtaA. We recently described two surface anchored proteins, ChtA and ChtC, which also bind hemin and Hb. ChtA and ChtC share structural similarities to HtaA; however, a function for ChtA and ChtC was not determined. In this study, we identified additional host iron sources that are utilized by C. diphtheriae. We show that several C. diphtheriae strains use the hemoglobin-haptoglobin (Hb-Hp) complex as an iron source. We report that an htaA deletion mutant of C. diphtheriae strain 1737 is unable to use the Hb-Hp complex as an iron source, and we further demonstrate that a chtA-chtC double mutant is also unable to use Hb-Hp iron. Single-deletion mutants of chtA or chtC use Hb-Hp iron in a manner similar to that of the wild type. These findings suggest that both HtaA and either ChtA or ChtC are essential for the use of Hb-Hp iron. Enzyme-linked immunosorbent assay (ELISA) studies show that HtaA binds the Hb-Hp complex, and the substitution of a conserved tyrosine (Y361) for alanine in HtaA results in significantly reduced binding. C. diphtheriae was also able to use human serum albumin (HSA) and myoglobin (Mb) but not hemopexin as iron sources. These studies identify a biological function for the ChtA and ChtC proteins and demonstrate that the use of the Hb-Hp complex as an iron source by C. diphtheriae requires multiple iron-regulated surface components.

  18. Outbreak investigation for toxigenic Corynebacterium diphtheriae wound infections in refugees from Northeast Africa and Syria in Switzerland and Germany by whole genome sequencing.

    PubMed

    Meinel, D M; Kuehl, R; Zbinden, R; Boskova, V; Garzoni, C; Fadini, D; Dolina, M; Blümel, B; Weibel, T; Tschudin-Sutter, S; Widmer, A F; Bielicki, J A; Dierig, A; Heininger, U; Konrad, R; Berger, A; Hinic, V; Goldenberger, D; Blaich, A; Stadler, T; Battegay, M; Sing, A; Egli, A

    2016-12-01

    Toxigenic Corynebacterium diphtheriae is an important and potentially fatal threat to patients and public health. During the current dramatic influx of refugees into Europe, our objective was to use whole genome sequencing for the characterization of a suspected outbreak of C. diphtheriae wound infections among refugees. After conventional culture, we identified C. diphtheriae using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and investigated toxigenicity by PCR. Whole genome sequencing was performed on a MiSeq Illumina with >70×coverage, 2×250 bp read length, and mapping against a reference genome. Twenty cases of cutaneous C. diphtheriae in refugees from East African countries and Syria identified between April and August 2015 were included. Patients presented with wound infections shortly after arrival in Switzerland and Germany. Toxin production was detected in 9/20 (45%) isolates. Whole genome sequencing-based typing revealed relatedness between isolates using neighbour-joining algorithms. We detected three separate clusters among epidemiologically related refugees. Although the isolates within a cluster showed strong relatedness, isolates differed by >50 nucleotide polymorphisms. Toxigenic C. diphtheriae associated wound infections are currently observed more frequently in Europe, due to refugees travelling under poor hygienic conditions. Close genetic relatedness of C. diphtheriae isolates from 20 refugees with wound infections indicates likely transmission between patients. However, the diversity within each cluster and phylogenetic time-tree analysis suggest that transmissions happened several months ago, most likely outside Europe. Whole genome sequencing offers the potential to describe outbreaks at very high resolution and is a helpful tool in infection tracking and identification of transmission routes.

  19. Culture-negative prosthetic valve endocarditis with concomitant septicemia due to a nontoxigenic Corynebacterium diphtheriae biotype gravis isolate in a patient with multiple risk factors.

    PubMed

    Clinton, Lani Kai; Bankowski, Matthew J; Shimasaki, Teppei; Sae-Ow, Wichit; Whelen, A Christian; O'Connor, Norman; Kim, Wesley; Young, Royden

    2013-11-01

    A 54-year-old female with a prosthetic mitral valve presented with a 3-day history of dizziness, subjective fever, and chills. Blood cultures were positive for a pleomorphic Gram-positive rod. Initial phenotypic testing could only support the identification of a Corynebacterium species. Nucleic acid sequencing (16S rRNA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) were conclusive for Corynebacterium diphtheriae. Definitive phenotypic testing classified the strain as nontoxigenic C. diphtheriae biotype Gravis.

  20. The designing of anti-diphtheria serotherapy at the Institut Pasteur (1888-1900): the role of a supranational network of microbiologists.

    PubMed

    Gachelin, Gabriel

    2007-01-01

    The development of anti-diphtheria serotherapy at the Institut Pasteur immediately follows the crisis known as the Pasteur-Koch debate. Research on diphtheria in Paris is indicative of the importance granted by Pasteurian scientists to Koch's school criticisms. After 1887, relations between French and German bacteriologists become more relaxed. A scientific and social network develops between them. It later extends to other fields of research at the Institut Pasteur, particularly therapeutic chemistry. The evolution of Franco-German relations at the Institut Pasteur is placed in the general framework of the way French universities considered German science.

  1. Development and validation of a multiplex immunoassay for the simultaneous determination of serum antibodies to Bordetella pertussis, diphtheria and tetanus.

    PubMed

    van Gageldonk, Pieter G M; van Schaijk, Frank G; van der Klis, Fiona R; Berbers, Guy A M

    2008-06-01

    To increase testing of vaccine induced humoral immunity in immune surveillance studies and vaccine trials, a rapid and simple microsphere-based multiplex assay (pentaplex) was developed for the quantitation of IgG serum antibodies directed against the Bordetella pertussis antigens: Pertussis Toxin (Ptx), Filamentous hemagglutinin (FHA), Pertactin (Prn) and to Diphtheria toxin and Tetanus toxin. All individual antigens were covalently linked to carboxylated microspheres. The method was validated with different serum panels (n=60-78 samples). With the Multiplex Immunoassay (MIA) no evidence for bead interference between monoplex and pentaplex was found. The specificity of the method was shown by a heterologous inhibition of <16% and homologous inhibition of >92%. The pentaplex MIA appeared sensitive with lower limits of quantitation (LLOQ) well below those for ELISA (enzyme-linked immuno-sorbant assay). Assay reproducibility was high with intra-assay variability less than 10% and inter-assay variability below 14%. The reproducibility of the bead conjugation was good and beads could be stored up to at least 6 months without quality reduction. Importantly, the correlation of the pentaplex MIA with the individual ELISAs was excellent, R>0.98 for the Pertussis antigens and R=0.95 for Diphtheria and R=0.98 for Tetanus. Serum IgG antibodies to B. pertussis, Diphtheria and Tetanus can be measured easily, specific and reproducible using the pentaplex MIA. The pentaplex MIA shares features of the ELISA with the additional advantages of high sample throughput and small sample volumes and antigen required.

  2. Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment.

    PubMed

    Draganova, Elizabeth B; Akbas, Neval; Adrian, Seth A; Lukat-Rodgers, Gudrun S; Collins, Daniel P; Dawson, John H; Allen, Courtni E; Schmitt, Michael P; Rodgers, Kenton R; Dixon, Dabney W

    2015-11-03

    The heme uptake pathway (hmu) of Corynebacterium diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type (WT) HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies indicates that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A is reduced readily in the presence of dithionite. The FeCO Raman shifts in WT, H136A, and Y235A HmuT-CO complexes provide further evidence of the axial ligand assignments. Additionally, these frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A-CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT.

  3. Cell surface display of minor pilin adhesins in the form of a simple heterodimeric assembly in Corynebacterium diphtheriae.

    PubMed

    Chang, Chungyu; Mandlik, Anjali; Das, Asis; Ton-That, Hung

    2011-03-01

    Pilus assembly in Gram-positive bacteria occurs by a two-step mechanism, whereby pilins are polymerized and then covalently anchored to the cell wall. In Corynebacterium diphtheriae, the pilin-specific sortase SrtA catalyses polymerization of the SpaA-type pilus, consisting of the shaft pilin SpaA, tip pilin SpaC and minor pilin SpaB. Cell wall anchoring of the SpaA polymers is triggered when SrtA incorporates SpaB into the pilus base via lysine-mediated transpeptidation; anchoring to the cell wall peptidoglycan is subsequently catalysed by the housekeeping sortase SrtF. Here we show that SpaB and SpaC formed a heterodimer independent of SpaA polymerization. SrtA was absolutely required for the formation of the SpaBC heterodimer, while SrtF facilitated the optimal cell wall anchoring of this heterodimer. Alanine substitution of the SpaB lysine residue K139 or truncation of the SpaB cell wall-sorting signal (CWSS) abolished assembly of the SpaBC heterodimer, hence underscoring SpaB function in transpeptidation and cell wall linkage. Importantly, sortase specificity for the cell wall-anchoring step was found to be dependent on the LAFTG motif within the SpaB CWSS. Thus, C. diphtheriae employs a common sortase-catalysed mechanism involving lysine-mediated transpeptidation to generate both adhesive pilus and simple heterodimeric structures on the bacterial the cell wall.

  4. Effect of skin barrier disruption on immune responses to topically applied cross-reacting material, CRM(197), of diphtheria toxin.

    PubMed

    Godefroy, S; Peyre, M; Garcia, N; Muller, S; Sesardic, D; Partidos, C D

    2005-08-01

    The high accessibility of the skin and the presence of immunocompetent cells in the epidermis makes this surface an attractive route for needle-free administration of vaccines. However, the lining of the skin by the stratum corneum is a major obstacle to vaccine delivery. In this study we examined the effect of skin barrier disruption on the immune responses to the cross-reacting material CRM(197), a nontoxic mutant of diphtheria toxin (DTx) that is considered as a vaccine candidate. Application of CRM(197), together with cholera toxin (CT), onto the tape-stripped skin of mice elicited antibody responses that had anti-DTx neutralizing activity. Vaccine delivery onto mildly ablated skin or intact skin did not elicit any detectable anti-CRM(197) antibodies. Mice immunized with CRM(197) alone onto the tape-stripped skin mounted a vigorous antigen-specific proliferative response. In contrast, the induction of cellular immunity after CRM(197) deposition onto mildly ablated or intact skin was adjuvant dependent. Furthermore, epidermal cells were activated and underwent apoptosis that was more pronounced when the stratum corneum was removed by tape stripping. Overall, these findings highlight the potential for transcutaneous delivery of CRM(197) and establish a correlation between the degree of barrier disruption and levels of antigen-specific immune responses. Moreover, these results provide the first evidence that the development of a transcutaneous immunization strategy for diphtheria, based on simple and practical methods to disrupt the skin barrier, is feasible.

  5. Immunogenicity of meningococcal quadrivalent (serogroup A, C, W135 and Y) tetanus toxoid conjugate vaccine: systematic review and meta-analysis.

    PubMed

    Pellegrino, Paolo; Perrone, Valentina; Radice, Sonia; Capuano, Annalisa; Clementi, Emilio

    2015-02-01

    Meningococcal meningitis represents one of the leading cause of bacterial meningitis in developed countries. Among the thirteen described serogroups, only five are usually responsible of invasive infections making immunisation against multiple serogroups the best strategy to protect individuals from this disease. Herein we carried out a systematic review and meta-analysis, in accordance with the PRISMA statement, of the recently EU-licensed meningococcal ACWY-tetanus toxoid conjugate vaccine (MenACWY-TT). We included 15 randomised clinical trials, comparing MenACWY-TT and Men-PS (ten studies), MenACWY-TT and MenC-CRM197 (four studies) and MenACWY-TT and MenACWY-DT (one study). All studies included in the meta-analysis showed high immunogenicity for MenACWY-TT vaccines in all tested serogroups. Our results suggest that the MenACWY-TT vaccine is as immunogenic as the other commercial available meningococcal vaccines.

  6. Corynebacterium diphtheriae 67-72p hemagglutinin, characterized as the protein DIP0733, contributes to invasion and induction of apoptosis in HEp-2 cells.

    PubMed

    Sabbadini, Priscila Soares; Assis, Maria Cristina; Trost, Eva; Gomes, Débora Leandro Rama; Moreira, Lilian Oliveira; Dos Santos, Cíntia Silva; Pereira, Gabriela Andrade; Nagao, Prescilla Emy; Azevedo, Vasco Ariston de Carvalho; Hirata Júnior, Raphael; Dos Santos, André Luis Souza; Tauch, Andreas; Mattos-Guaraldi, Ana Luíza

    2012-03-01

    Although Corynebacterium diphtheriae has been classically described as an exclusively extracellular pathogen, there is growing evidence that it may be internalized by epithelial cells. The aim of the present report was to investigate the nature and involvement of the surface-exposed non-fimbrial 67-72 kDa proteins (67-72p), previously characterized as adhesin/hemagglutinin, in C. diphtheriae internalization by HEp-2 cells. Transmission electron microscopy and bacterial internalization inhibition assays indicated the role of 67-72p as invasin for strains of varied sources. Cytoskeletal changes with accumulation of polymerized actin in HEp-2 cells beneath adherent 67-72p-adsorbed microspheres were observed by the Fluorescent actin staining test. Trypan blue staining method and Methylthiazole tetrazolium reduction assay showed a significant decrease in viability of HEp-2 cells treated with 67-72p. Morphological changes in HEp-2 cells observed after treatment with 67-72p included vacuolization, nuclear fragmentation and the formation of apoptotic bodies. Flow cytometry revealed an apoptotic volume decrease in HEp-2 cells treated with 67-72p. Moreover, a double-staining assay using Propidium Iodide/Annexin V gave information about the numbers of vital vs. early apoptotic cells and late apoptotic or secondary necrotic cells. The comparative analysis of MALDI-TOF MS experiments with the probes provided for 67-72p CDC-E8392 with an in silico proteome deduced from the complete genome sequence of C. diphtheriae identified with significant scores 67-72p as the protein DIP0733. In conclusion, DIP0733 (67-72p) may be directly implicated in bacterial invasion and apoptosis of epithelial cells in the early stages of diphtheria and C. diphtheriae invasive infection.

  7. HtaA is an iron-regulated hemin binding protein involved in the utilization of heme iron in Corynebacterium diphtheriae.

    PubMed

    Allen, Courtni E; Schmitt, Michael P

    2009-04-01

    Many human pathogens, including Corynebacterium diphtheriae, the causative agent of diphtheria, use host compounds such as heme and hemoglobin as essential iron sources. In this study, we examined the Corynebacterium hmu hemin transport region, a genetic cluster that contains the hmuTUV genes encoding a previously described ABC-type hemin transporter and three additional genes, which we have designated htaA, htaB, and htaC. The hmu gene cluster is composed of three distinct transcriptional units. The htaA gene appears to be part of an iron- and DtxR-regulated operon that includes hmuTUV, while htaB and htaC are transcribed from unique DtxR-regulated promoters. Nonpolar deletion of either htaA or the hmuTUV genes resulted in a reduced ability to use hemin as an iron source, while deletion of htaB had no effect on hemin iron utilization in C. diphtheriae. A comparison of the predicted amino acid sequences of HtaA and HtaB showed that they share some sequence similarity, and both proteins contain leader sequences and putative C-terminal transmembrane regions. Protein localization studies with C. diphtheriae showed that HtaA is associated predominantly with the cell envelope when the organism is grown in minimal medium but is secreted during growth in nutrient-rich broth. HtaB and HmuT were detected primarily in the cytoplasmic membrane fraction regardless of the growth medium. Hemin binding studies demonstrated that HtaA and HtaB are able to bind hemin, suggesting that these proteins may function as cell surface hemin receptors in C. diphtheriae.

  8. Single radial immunodiffusion as a method for the assay of the acellular pertussis vaccine components, pertussis toxoid, filamentous haemagglutinin and pertactin.

    PubMed

    Xing, D K; Canthaboo, C; Corbel, M J; Schild, G C

    1998-09-01

    The development of acellular pertussis vaccines has raised a number of issues relevant to the control of these products. Of particular importance is the need for robust and accurate in vitro assays for the antigen content of the vaccines which might contain up to five different antigen components, each of which needs to be independently assayed. This paper describes a simple method for the quantification of three component antigens. Because relatively high doses of purified antigens are used in those preparations, the elimination of residual toxicity is a major concern. This is achieved by genetic modification of chemical treatment. The latter results in modification of the immunological reactivity of the antigens making direct assay by such methods as ELISA ineffective. A single radial diffusion technique using polyclonal antisera for the assay of pertussis toxoid (PTxd), chemically treated filamentous haemagglutinin (FHA) and pertactin (69 kDa) has been developed. The method uses low concentrations of antisera, allowing accurate and reproducible quantification of antigen content as low as 25 microg/ml of protein for pertussis toxoid and filamentous haemagglutinin and 5 microg/ml for pertactin. Since by the addition of detergent, diffusible subunits are produced irrespective of the original physical state of the antigens, the assay is suitable for assay of these antigens after detoxification/or stabilization by chemical treatment and is able to determine the differences between preparations which have the same protein concentration but different antigenic contents. This provides a means for assuring the consistency of the antigens after detoxification/or chemical stabilization which could be used as an in-process control method for acellular pertussis vaccines.

  9. Immunogenicity of a low-dose diphtheria, tetanus and acellular pertussis combination vaccine with either inactivated or oral polio vaccine compared to standard-dose diphtheria, tetanus, acellular pertussis when used as a pre-school booster in UK children: A 5-year follow-up of a randomised controlled study.

    PubMed

    John, T; Voysey, M; Yu, L M; McCarthy, N; Baudin, M; Richard, P; Fiquet, A; Kitchin, N; Pollard, A J

    2015-08-26

    This serological follow up study assessed the kinetics of antibody response in children who previously participated in a single centre, open-label, randomised controlled trial of low-dose compared to standard-dose diphtheria booster preschool vaccinations in the United Kingdom (UK). Children had previously been randomised to receive one of three combination vaccines: either a combined adsorbed tetanus, low-dose diphtheria, 5-component acellular pertussis and inactivated polio vaccine (IPV) (Tdap-IPV, Repevax(®); Sanofi Pasteur MSD); a combined adsorbed tetanus, low-dose diphtheria and 5-component acellular pertussis vaccine (Tdap, Covaxis(®); Sanofi Pasteur MSD) given concomitantly with oral polio vaccine (OPV); or a combined adsorbed standard-dose diphtheria, tetanus, 2-component acellular pertussis and IPV (DTap-IPV, Tetravac(®); Sanofi Pasteur MSD). Blood samples for the follow-up study were taken at 1, 3 and 5 years after participation in the original trial (median, 5.07 years of age at year 1), and antibody persistence to each vaccine antigen measured against defined serological thresholds of protection. All participants had evidence of immunity to diphtheria with antitoxin concentrations greater than 0.01IU/mL five years after booster vaccination and 75%, 67% and 79% of children who received Tdap-IPV, Tdap+OPV and DTap-IPV, respectively, had protective antitoxin levels greater than 0.1IU/mL. Long lasting protective immune responses to tetanus and polio antigens were also observed in all groups, though polio responses were lower in the sera of those who received OPV. Low-dose diphtheria vaccines provided comparable protection to the standard-dose vaccine and are suitable for use for pre-school booster vaccination.

  10. Effect of total lymphoid irradiation on levels of serum autoantibodies in systemic lupus erythematosus and in rheumatoid arthritis

    SciTech Connect

    Tanay, A.; Schiffman, G.; Strober, S.

    1986-01-01

    The effects of total lymphoid irradiation (TLI) on serum levels of autoantibodies, and of antibodies to diphtheria toxoid, tetanus toxoid, and pneumococcal polysaccharide in patients with lupus nephritis were compared with those previously observed in rheumatoid arthritis (RA) patients. Baseline levels of antibodies to diphtheria toxoid and tetanus toxoid decreased significantly after TLI in patients with lupus and RA, but antibody levels to pneumococcal polysaccharide remained unchanged. After TLI, the levels of antinuclear and anti-DNA antibodies were reduced significantly in lupus, but levels of rheumatoid factor, antinuclear, and antigranulocyte antibodies all tended to increase in RA.

  11. Structure of the response regulator ChrA in the haem-sensing two-component system of Corynebacterium diphtheriae.

    PubMed

    Doi, Akihiro; Nakamura, Hiro; Shiro, Yoshitsugu; Sugimoto, Hiroshi

    2015-08-01

    ChrA is a response regulator (RR) in the two-component system involved in regulating the degradation and transport of haem (Fe-porphyrin) in the pathogen Corynebacterium diphtheriae. Here, the crystal structure of full-length ChrA is described at a resolution of 1.8 Å. ChrA consists of an N-terminal regulatory domain, a long linker region and a C-terminal DNA-binding domain. A structural comparison of ChrA with other RRs revealed substantial differences in the relative orientation of the two domains and the conformation of the linker region. The structural flexibility of the linker could be an important feature in rearrangement of the domain orientation to create a dimerization interface to bind DNA during haem-sensing signal transduction.

  12. Systemic antibody responses induced by a two-component Clostridium difficile toxoid vaccine protect against C. difficile-associated disease in hamsters.

    PubMed

    Anosova, Natalie G; Brown, Anna M; Li, Lu; Liu, Nana; Cole, Leah E; Zhang, Jinrong; Mehta, Hersh; Kleanthous, Harry

    2013-09-01

    Clostridium difficile infection (CDI) has been identified as the leading cause of nosocomial diarrhoea and pseudomembranous colitis associated with antibiotic therapy. Recent epidemiological changes as well as increases in the number of outbreaks of strains associated with increased virulence and higher mortality rates underscore the importance of identifying alternatives to antibiotics to manage this important disease. Animal studies have clearly demonstrated the roles that toxins A and B play in gut inflammation as well as diarrhoea; therefore it is not surprising that serum anti-toxin A and B IgG are associated with protection against recurrent CDI. In humans, strong humoral toxin-specific immune responses elicited by natural C. difficile infection is associated with recovery and lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. The first generation of C. difficile vaccine that contained inactivated toxin A and B was found to be completely protective against death and diarrhoea in the hamster C. difficile challenge model. When tested in young healthy volunteers in Phase I clinical trials, this investigational vaccine was shown to be safe and immunogenic. Moreover, in a separate study this vaccine was able to prevent further relapses in three out of three patients who had previously suffered from chronic relapsing C. difficile-associated diarrhoea. Herein we examined the immunogenicity and protective activity of a next-generation Sanofi Pasteur two-component highly purified toxoid vaccine in a C. difficile hamster model. This model is widely recognized as a stringent and relevant choice for the evaluation of novel treatment strategies against C. difficile and was used in preclinical testing of the first-generation vaccine candidate. Intramuscular (i.m.) immunizations with increasing doses of this adjuvanted toxoid vaccine protected hamsters from mortality and disease symptoms in a dose-dependent manner. ELISA

  13. Self-reported history of vaccination and disease and immunity against hepatitis A, hepatitis B, tetanus, diphtheria and varicella among Spanish military recruits.

    PubMed

    Arteaga, Alejandro; Desviat, Pilar Vallejo; Jaqueti, Jeronimo; Santos, Juana; de Miguel, Angel Gil; Garcia, Rodrigo Jiménez

    2010-02-01

    This study aims to evaluate the immune status against hepatitis A, hepatitis B, tetanus, diphtheria and varicella in military recruits and the validity of self-reporting of their disease and vaccination history. A total of 226 participants were studied (mean age, 20.2 years; SD 1.7). 10.4% presented antibodies to hepatitis A, 78.3% to hepatitis B, 94.2% to tetanus, 77.4% to diphtheria and 81.9% to varicella. The relationship between self-reporting of vaccination history and seroprotection showed a high Positive Predictive Value for tetanus (98.8%) and a high Negative Predictive Value for hepatitis A (91%). Hepatitis A vaccination and serology testing for varicella and Hepatitis B on joining the Spanish armed forces are recommended.

  14. Measuring immunoglobulin g antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin with single-antigen enzyme-linked immunosorbent assays and a bead-based multiplex assay.

    PubMed

    Reder, Sabine; Riffelmann, Marion; Becker, Christian; Wirsing von König, Carl Heinz

    2008-05-01

    Bead-based assay systems offer the possibility of measuring several specific antibodies in one sample simultaneously. This study evaluated a vaccine panel of a multianalyte system that measures antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin (PT) from Bordetella pertussis. The antibody concentrations of human immunoglobulin G (IgG) to PT, tetanus toxin, and diphtheria toxin were measured in 123 serum pairs (total of 246 sera) from a vaccine study. The multianalyte bead assay was compared to a standardized in-house IgG- anti-PT enzyme-linked immunosorbent assay (ELISA) of the German reference laboratory for bordetellae, as well as to various commercially available ELISAs for anti-PT IgG, anti-tetanus IgG, and anti-diphtheria IgG. The results of the multiplex assay regarding the antibodies against diphtheria toxin compared favorably with a regression coefficient of 0.938 for values obtained with an ELISA from the same manufacturer used as a reference. Similarly, antibodies to tetanus toxin showed a correlation of 0.910 between the reference ELISA and the multianalyte assay. A correlation coefficient of 0.905 was found when an "in-house" IgG anti-PT and the multiplex assay were compared. Compared to single ELISA systems from two other manufacturers, the multiplex assay performed similarly well or better. The multianalyte assay system was a robust system with fast and accurate results, analyzing three parameters simultaneously in one sample. The system was well suited to quantitatively determine relevant vaccine induced antibodies compared to in-house and commercially available single-antigen ELISA systems.

  15. Expression and Immunogenicity of a Mutant Diphtheria Toxin Molecule, CRM197, and Its Fragments in Salmonella typhi Vaccine Strain CVD 908-htrA

    PubMed Central

    Orr, Nadav; Galen, James E.; Levine, Myron M.

    1999-01-01

    Mutant diphtheria toxin molecule CRM197 and fragments thereof were expressed in attenuated Salmonella typhi CVD 908-htrA, and the constructs were tested for their ability to induce serum antitoxin. Initially, expressed proteins were insoluble, and the constructs failed to induce neutralizing antitoxin. Soluble CRM197 was expressed at low levels by utilizing the hemolysin A secretion system from Escherichia coli. PMID:10417208

  16. A slow-forming isopeptide bond in the structure of the major pilin SpaD from Corynebacterium diphtheriae has implications for pilus assembly

    SciTech Connect

    Kang, Hae Joo; Paterson, Neil G.; Kim, Chae Un; Middleditch, Martin; Chang, Chungyu; Ton-That, Hung; Baker, Edward N.

    2014-05-01

    Two crystal structures of the major pilin SpaD from C. diphtheriae have been determined at 1.87 and 2.5 Å resolution. The N-terminal domain is found to contain an isopeptide bond that forms slowly over time in the recombinant protein. Given its structural context, this provides insight into the relationship between internal isopeptide-bond formation and pilus assembly. The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed by C. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys–Asn isopeptide bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to bond formation. Two crystal structures, of the full-length three-domain protein at 2.5 Å resolution and of a two-domain (D2-D3) construct at 1.87 Å resolution, show that each of the three Ig-like domains contains a single Lys–Asn isopeptide-bond cross-link, assumed to give mechanical stability as in other such pili. Additional stabilizing features include a disulfide bond in the D3 domain and a calcium-binding loop in D2. The N-terminal D1 domain is more flexible than the others and, by analogy with other major pilins of this type, the slow formation of its isopeptide bond can be attributed to its location adjacent to the lysine used in sortase-mediated polymerization during pilus assembly.

  17. A thiol-disulfide oxidoreductase of the Gram-positive pathogen Corynebacterium diphtheriae is essential for viability, pilus assembly, toxin production and virulence.

    PubMed

    Reardon-Robinson, Melissa E; Osipiuk, Jerzy; Jooya, Neda; Chang, Chungyu; Joachimiak, Andrzej; Das, Asis; Ton-That, Hung

    2015-12-01

    The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.

  18. Study on the use of an enzyme-linked immunosorbent assay in determining human antibodies to diphtheria toxin as compared with a reference toxin neutralization assay.

    PubMed

    Skoura, L; Efstratiou, A; Tsakris, A; Pournaras, S; George, R C; Douboyas, J

    1999-07-01

    Serum samples from 156 Greek persons were assessed by an IgG-specific enzyme-linked immunosorbent assay (ELISA) and a reference tissue culture toxin-neutralization (TN) assay for the quantitation of diphtheria toxin antibodies. By the reference method, 7.7% of the persons were susceptible to diphtheria (antitoxin < 0.01 IU/ml), 28.8% had basic protection (antitoxin 0.01-0.09 IU/ml) and 63.5% were fully protective (antitoxin > or = 0.1 IU/ ml), while the corresponding figures were 17.9, 36.5 and 45.5% when they were tested by the immunoassay. None of the samples been susceptible by the TN assay were found to have some protection when tested by ELISA. However, three (6.7%) of the 45 samples showing a basic protection with TN, were fully protective when titrated by the immunoassay. In addition, 31 (31.3%) of the 99 samples been fully protective by the bioassay, were found to be either basically protective or susceptible by means of the ELISA. Overall, validity features of the immunoassay were: sensitivity 68.7%, specificity 94.7%, positive predictive value 95.8% and negative predictive value 63.5%. The ELISA tested in our study could be used to determine diphtheria antitoxin in individuals needed a booster immunization (susceptible or basic protective samples), although it might falsely include in the above categories samples that are within the fully protective levels of antibodies.

  19. Definition of Human Epitopes Recognized in Tetanus Toxoid and Development of an Assay Strategy to Detect Ex Vivo Tetanus CD4+ T Cell Responses

    PubMed Central

    da Silva Antunes, Ricardo; Paul, Sinu; Sidney, John; Weiskopf, Daniela; Dan, Jennifer M.; Phillips, Elizabeth; Mallal, Simon; Crotty, Shane; Sette, Alessandro; Lindestam Arlehamn, Cecilia S.

    2017-01-01

    Despite widespread uses of tetanus toxoid (TT) as a vaccine, model antigen and protein carrier, TT epitopes have been poorly characterized. Herein we defined the human CD4+ T cell epitope repertoire by reevaluation of previously described epitopes and evaluation of those derived from prediction of HLA Class II binding. Forty-seven epitopes were identified following in vitro TT stimulation, with 28 epitopes accounting for 90% of the total response. Despite this diverse range of epitopes, individual responses were associated with only a few immunodominant epitopes, with each donor responding on average to 3 epitopes. For the top 14 epitopes, HLA restriction could be inferred based on HLA typing of the responding donors. HLA binding predictions re-identified the vast majority of known epitopes, and identified 24 additional novel epitopes. With these epitopes, we created a TT epitope pool, which allowed us to characterize TT responses directly ex vivo using a cytokine-independent Activation Induced Marker (AIM) assay. These TT responses were highly Th1 or Th2 polarized, which was dependent upon the original priming vaccine, either the cellular DTwP or acellular DTaP formulation. This polarization remained despite the original priming having occurred decades past and a recent booster immunization with a reduced acellular vaccine formulation. While TT responses following booster vaccination were not durably increased in magnitude, they were associated with a relative expansion of CD4+ effector memory T cells. PMID:28081174

  20. Hollow-fire bioreactors compared to batch and chemostat culture for the production of a recombinant toxoid by a marine Iibrio.

    PubMed

    Lloyd, J R; Hirst, T R; Bunch, A W

    1997-08-01

    Bioreactor selection is important for maximising the productivity of recombinant organisms. In this paper a comparison is made between growth and recombinant protein synthesis in three types of bioreactor containing a marine Vibrio capable of heterologous expression and secretion of the non-toxic B-subunit pentamer of Escherichia coli heat-labile enterotoxin, EtxB. The heterologous gene was located on the plasmid pMMB68. Resistance to carbenicillin was used to select for plasmid-containing cells. In batch and continuous culture. Volumetric productivities were highest when cells were grown in the presence of carbenicillin. Without antibiotic selection, the highest volumetric productivity (9.4 mg EtxB-1 h-1) was observed in hollow-fibre bioreactors, and the production phase could be maintained for over 50 h. The highest specific productivity under these conditions was found in batch culture, but the maximal production phase was only of 5 h duration. In hollow-fibre reactors the type of fibre used significantly affected productivity, both with regards to the maintenance of reactor integrity and by allowing passage of the recombinant toxoid through the selectively permeable membrane. Where contamination of the product with carbenicillin is to be avoided, these bioreactors are superior to batch or continuous culture.

  1. PLGA nano/micro particles encapsulated with pertussis toxoid (PTd) enhances Th1/Th17 immune response in a murine model.

    PubMed

    Li, Pan; Asokanathan, Catpagavalli; Liu, Fang; Khaing, Kyi Kyi; Kmiec, Dorota; Wei, Xiaoqing; Song, Bing; Xing, Dorothy; Kong, Deling

    2016-11-20

    Poly(lactic-co-glycolic acid) (PLGA) based nano/micro particles were investigated as a potential vaccine platform for pertussis antigen. Presentation of pertussis toxoid as nano/micro particles (NP/MP) gave similar antigen-specific IgG responses in mice compared to soluble antigen. Notably, in cell line based assays, it was found that PLGA based nano/micro particles enhanced the phagocytosis of fluorescent antigen-nano/micro particles by J774.2 murine monocyte/macrophage cells compared to soluble antigen. More importantly, when mice were immunised with the antigen-nano/micro particles they significantly increased antigen-specific Th1 cytokines INF-γ and IL-17 secretion in splenocytes after in vitro re-stimulation with heat killed Bordetalla pertussis, indicating the induction of a Th1/Th17 response. Also, presentation of pertussis antigen in a NP/MP formulation is able to provide protection against respiratory infection in a murine model. Thus, the NP/MP formulation may provide an alternative to conventional acellular vaccines to achieve a more balanced Th1/Th2 immune response.

  2. Autoantibody response and pregnancy-related pathology induced by combined LPS and tetanus toxoid hyperimmunization in BALB/c and C57BL/6 mice.

    PubMed

    Petrušić, Vladimir; Todorović, Nevena; Živković, Irena; Dimitrijević, Rajna; Muhandes, Lina; Rajnpreht, Irena; Dimitrijević, Ljiljana

    2015-03-01

    Recent data concerning antiphospholipid syndrome (APS) induction have shown that β2-glycoprotein I (β2GPI) binds lipopolysaccharide (LPS), which results in conformational changes, exposition of a cryptic epitope and possible pathological anti-β2GPI antibody production. In order to investigate the effects of LPS on the induction of APS-related pathology, we performed hyperimmunization of BALB/c and C57BL/6 mice with LPS, alone or in combination with tetanus toxoid (TTd), a protein structurally similar to β2GPI. We report that, although high affinity pathological anti-β2GPI antibodies were produced in all groups of animals, the reproductive pathology was recorded only in mice that received both LPS and TTd, implying on the important roles of both infections and molecular mimicry in APS pathogenesis. Moreover, APS-related reproductive pathology was more pronounced in BALB/c (lowered fertility and fecundity) than C57BL/6 mice (lowered fecundity), which correlated well with the disruption in natural antibody network observed in BALB/c mouse strain.

  3. Determinants of utilization of sufficient tetanus toxoid immunization during pregnancy: evidence from the Kenya Demographic and Health Survey, 2008-2009.

    PubMed

    Haile, Zelalem T; Chertok, Ilana R Azulay; Teweldeberhan, Asli K

    2013-06-01

    Although the effectiveness of tetanus toxoid (TT) immunization during pregnancy in preventing maternal and neonatal tetanus is well established, in many developing countries, TT immunization programs are underutilized. The objective of this study was to examine factors associated with sufficient TT immunization among postpartum women in Kenya. Population based secondary data analysis was conducted using de-identified data from the 2008-2009 Kenyan Demographic and Health Survey (KDHS) for 1,370 female participants who had a live birth during or within 12 months of the cross-sectional survey. Chi-square test and independent sample t test were conducted to assess bivariate associations and a multivariable logistic regression analysis was conducted to examine associations before and after adjustment for demographic, socioeconomic, cultural, and access to care factors. The main factors contributing to having been sufficiently immunized against tetanus were lower birth order, higher household wealth index, women's employment, making joint health-related decisions with a partner, and higher number of antenatal care visits. Implications for health care providers and other professionals involved in development of strategies and interventions aimed at improving immunization rates are discussed.

  4. Enhanced mucosal immune responses against tetanus toxoid using novel delivery system comprised of chitosan-functionalized gold nanoparticles and botanical adjuvant: characterization, immunogenicity, and stability assessment.

    PubMed

    Barhate, Ganesh; Gautam, Manish; Gairola, Sunil; Jadhav, Suresh; Pokharkar, Varsha

    2014-11-01

    Approaches based on combined use of delivery systems and adjuvants are being favored to maximize efficient mucosal delivery of antigens. Here, we describe a novel delivery system comprised of chitosan-functionalized gold nanoparticles (CsAuNPs) and saponin-containing botanical adjuvant; Asparagus racemosus extract (ARE) for oral delivery of tetanus toxoid (TT). A significant increase in TT-specific IgG (34.53-fold) and IgA (43.75-fold) was observed when TT-CsAuNPs were formulated with ARE (TT-ARE-CsAuNPs). The local IgA immune responses for TT also showed a significant increase (106.5-fold in intestine washes and 99.74-fold in feces) with ARE-based formulations as compared with plain TT group. No effect of ARE was observed on size, charge, and loading properties of CsAuNPs. Additionally, no effect of ARE and CsAuNPs was observed on antigenicity and secondary structure of TT as determined by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. The stability studies demonstrated excellent stability profile of formulation at recommended storage conditions. The study establishes the possible role of immunomodulatory adjuvants in particulate delivery systems for mucosal delivery of vaccines.

  5. Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development.

    PubMed

    Gupta, Neha; Shrestha, Abhinav; Panda, Amulya Kumar; Gupta, Satish Kumar

    2013-07-01

    Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and β-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 μg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine.

  6. Subnormal and waning immunity to tetanus toxoid in previously vaccinated HIV-infected children and response to booster doses of the vaccine.

    PubMed

    Choudhury, Shahana A; Matin, Fazle

    2013-12-01

    Little is known regarding waning immunity to tetanus toxoid (TT) in HIV-infected children and the need for booster doses before the recommended interval of 5-10 years. Anti-tetanus antibodies were assessed by ELISA in 24 HIV-infected and 24 control children. A protective level (>0.1 IU/ml) of TT antibodies was observed in 62% of HIV-infected children and in 100% of controls. HIV-infected children with five doses had a significantly (p=0.01) lower prevalence of protective immunity compared to controls. Follow-up anti-TT antibody levels in nine HIV-infected children declined from 1.27 to 0.26 IU/ml, but levels did not decline in the seven controls; five of the seven (71%) children with a non-protective level of antibodies responded with a level>0.16 IU/ml following one booster dose of the vaccine. HIV-infected children may need TT boosters before the recommended 5-10 years.

  7. Adjuvant dependence of APS pathology-related low-affinity antibodies during secondary immune response to tetanus toxoid in BALB/c mice.

    PubMed

    Zivković, Irena; Petrušić, Vladimir; Dimitrijević, Rajna; Stojanović, Marijana; Dimitrijević, Ljiljana

    2013-05-01

    One of the established animal models for autoimmune disease antiphospholipid syndrome (APS) is TTd hyperimmunization of mice. Tetanus toxoid (TTd) and plasma protein β2GPI share structural homology so that immunization with TTd induces appearance of cross-reactive antibodies. In this paper, we have investigated the presence and dynamic of fluctuation of specific (anti-TTd) and auto (anti-β2GPI) antibodies induced in BALB/c mice during secondary immune response after TTd immunization with alhydrogel or glycerol as adjuvants. In addition, we followed the induced reproductive pathology as a sign of autoimmune outcome. We show undoubtedly adjuvant dependance of (1) level of induced anti-TTd IgG antibodies, (2) changes in levels of low-affinity anti-β2GPI IgG antibodies, and (3) change in fecundity and fertility during secondary immune response. These findings once more indicate the importance of chosen adjuvants used for successful immunization and eventual autoantibody outcome, this time associated with the processes involving low affinity, natural antibodies.

  8. Immunogenicity of porcine P[6], P[7]-specific △VP8* rotavirus subunit vaccines with a tetanus toxoid universal T cell epitope.

    PubMed

    Wen, Xiaobo; Wei, Xiaoman; Ran, Xuhua; Ni, Hongbo; Cao, Si; Zhang, Yao

    2015-08-26

    Currently, commercial porcine rotavirus vaccines remain varied limitations. The objective of this study is to develop an alternative porcine rotavirus subunit vaccine candidate by parenteral administration, which enables to elicit robust immune responses against most prevalence porcine rotavirus strains. The bacterially-expressed porcine rotavirus P[6]- or P[7]-specific truncated VP8* (aa 64-223) recombinant protein with or without a universal tetanus toxoid CD4(+) T cell epitope P2 was generated. All the recombinant subunit proteins △VP8*s or P2-△VP8*s were of high solubility and high yields. The immunogenicity of each purified △VP8* and P2-△VP8* was evaluated in mice (10 μg/dose) or guinea pigs (20 μg/dose) immunized IM with 600 μg aluminum hydroxide three times at 2-week interval. The introduction of P2T cell epitope to P[7]-△VP8* elicited significantly higher IgG titer in mice than its absence. Comparatively, P2 epitope slightly enhanced the immunogenicity of P[6]-△VP8*. P2-P[7]△VP8* elicited high titer of neutralizing antibody against heterotypic P[7]-specific rotaviruses with varied G type combination. Our data indicated that two subunit vaccines could be plausible bivalent rotavirus vaccine candidate to provide antigenic coverage of porcine rotavirus strains of global or regional importance.

  9. Identification and isolation of kidney-derived stem cells from transgenic rats with diphtheria toxin-induced kidney damage

    PubMed Central

    Liu, Qing-Zhen; Chen, Xu-Dong; Liu, Gang; Guan, Guang-Ju

    2016-01-01

    Adult stem cells have been well characterized in numerous organs, with the exception of the kidneys. Therefore, the present study aimed to identify and isolate kidney-derived stem cells. A total of 12 Fischer 344 transgenic rats expressing the human diphtheria toxin receptor in podocyte cells of the kidney, were used in the present study. The rats were administered 5-bromo-2′-deoxyuridine (BrdU) in order to detect cellular proliferation. After 60 days, the rats were treated with the diphtheria toxin (DT), in order to induce kidney injury. Immunohistochemical analysis indicated that the number of BrdU-positive cells were increased following DT treatment. In addition, the expression of octamer-binding transcription factor 4 (Oct-4), a stem cell marker, was detected and suggested that kidney-specific stem cells were present in the DT-treated tissue samples. Furthermore, tissue samples exhibited repair of the DT-induced injury. Further cellular culturing was conducted in order to isolate the kidney-specific stem cells. After 5 weeks of culture, the majority of the cells were non-viable, with the exception of certain specialized, unique cell types, which were monomorphic and spindle-shaped in appearance. The unique cells were isolated and subjected to immunostaining and reverse transcription-polymerase chain reaction analyses in order to reconfirm the expression of Oct-4 and to detect the expression of Paired box 2 (Pax-2), which is necessary for the formation of kidney structures. The unique cells were positive for Oct-4 and Pax-2; thus suggesting that the identified cells were kidney-derived stem cells. The results of the present study suggested that the unique cell type identified in the kidneys of the DT-treated rats were kidney-specific stem cells that may have been involved in the repair of DT-induced tissue injury. In addition, these cells may provide a useful cell line for studying the fundamental characteristics of kidney stem cells, as well as identifying

  10. A diphtheria toxin resistance marker for in vitro and in vivo selection of stably transduced human cells.

    PubMed

    Picco, Gabriele; Petti, Consalvo; Trusolino, Livio; Bertotti, Andrea; Medico, Enzo

    2015-09-30

    We developed a selectable marker rendering human cells resistant to Diphtheria Toxin (DT). The marker (DT(R)) consists of a primary microRNA sequence engineered to downregulate the ubiquitous DPH2 gene, a key enzyme for the biosynthesis of the DT target diphthamide. DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth. DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers. As mice are insensitive to DT, DT(R)-based selection can be also applied in vivo. Direct injection of a GFP-DT(R) lentiviral vector into human cancer cell-line xenografts and patient-derived tumorgrafts implanted in mice, followed by systemic DT administration, yielded tumors entirely composed of permanently transduced cells and detectable by imaging systems. This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.

  11. ADP-ribosylation of translation elongation factor 2 by diphtheria toxin in yeast inhibits translation and cell separation.

    PubMed

    Mateyak, Maria K; Kinzy, Terri Goss

    2013-08-23

    Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADP(R)) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADP(R)-eEF2 to examine the effects of DT in vivo. ADP(R) of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in translation elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADP(R)-eEF2.

  12. Improved ELISA for determination of anti-diphtheria and/or anti-tetanus antitoxin antibodies in sera.

    PubMed

    Kristiansen, M; Aggerbeck, H; Heron, I

    1997-11-01

    Double-antigen ELISAs for detection and quantification of anti-tetanus or anti-diphtheria antibodies in serum have been developed. The assays showed good correlations with established toxin neutralizing assays and were functionally specific for IgG antibodies. The double-antigen set-up allows specific antibodies to bind to antigen-coated microtitre wells with one arm and the free arm to bind to biotin-labelled antigen. The amount of antibodies able to bind labelled antigen was assessed by adding enzyme-conjugated streptavidin and colour substrate followed by measurement of the colour using an ELISA reader. The double-antigen principle makes it possible to compare samples of different species on the same plate, permitting the direct use of existing international references of animal or human origin. The double-antigen ELISAs showed a detection limit of 0.00002 IU/ml for both antibodies and were suitable for quantifying antibodies in blood samples collected on filter paper as well as in serum. The assays required no special equipment compared to traditional ELISA.

  13. Morphometric evidence from C-synapses for phased Nissl body response in alpha-motoneurones retrogradely intoxicated with diphtheria toxin.

    PubMed

    Pullen, A H

    1990-02-12

    Diphtheria toxin (DTX) kills cells by inactivating ribosomal translocation and when used to retrogradely intoxicate cat intercostal motoneurones produces marked morphological alterations in Nissl bodies, including those specifically sited postsynaptic to C-type axon terminals. Here, qualitative examinations of 'intoxicated' postsynaptic Nissl bodies reveal a progressive structural alteration marked by rER dilatation, rER lamellae fragmentation but retention of both the highly ordered multilamellate organization and ribosomal attachment until final stages of Nissl body dissolution. Morphometric results identified 3 broad phases to the postintoxication response which differed in the degree of rER cisternal dilation, and the numerical and spatial relationships between rER-lamellae, rER-bound ribosomes and rER-associated polyribosomes. These phases reflect the known molecular basis of diphtheritic toxicity and contrast with the fast developing Nissl body reaction associated with the neurotoxin ricin which also invokes ribosomal dysfunction and has been used to mimic certain features of motor neurone disease. The cytopathology of DTX and ricin are compared in the Discussion.

  14. pH-triggered conformational switching of the diphtheria toxin T-domain: the roles of N-terminal histidines.

    PubMed

    Kurnikov, Igor V; Kyrychenko, Alexander; Flores-Canales, Jose C; Rodnin, Mykola V; Simakov, Nikolay; Vargas-Uribe, Mauricio; Posokhov, Yevgen O; Kurnikova, Maria; Ladokhin, Alexey S

    2013-08-09

    pH-induced conformational switching is essential for functioning of diphtheria toxin, which undergoes a membrane insertion/translocation transition triggered by endosomal acidification as a key step of cellular entry. In order to establish the sequence of molecular rearrangements and side-chain protonation accompanying the formation of the membrane-competent state of the toxin's translocation (T) domain, we have developed and applied an integrated approach that combines multiple techniques of computational chemistry [e.g., long-microsecond-range, all-atom molecular dynamics (MD) simulations; continuum electrostatics calculations; and thermodynamic integration (TI)] with several experimental techniques of fluorescence spectroscopy. TI calculations indicate that protonation of H257 causes the greatest destabilization of the native structure (6.9 kcal/mol), which is consistent with our early mutagenesis results. Extensive equilibrium MD simulations with a combined length of over 8 μs demonstrate that histidine protonation, while not accompanied by the loss of structural compactness of the T-domain, nevertheless results in substantial molecular rearrangements characterized by the partial loss of secondary structure due to unfolding of helices TH1 and TH2 and the loss of close contact between the C- and N-terminal segments. The structural changes accompanying the formation of the membrane-competent state ensure an easier exposure of the internal hydrophobic hairpin formed by helices TH8 and TH9, in preparation for its subsequent transmembrane insertion.

  15. Risk Factors of Delay Proportional Probability in Diphtheria-tetanus-pertussis Vaccination of Iranian Children; Life Table Approach Analysis.

    PubMed

    Mokhtari, Mohsen; Rezaeimanesh, Masoomeh; Mohammadbeigi, Abolfazl; Zahraei, Seyed Mohsen; Mohammadsalehi, Narges; Ansari, Hossein

    2015-01-01

    Despite success in expanded program immunization for an increase in vaccination coverage in the children of world, timeliness and schedule of vaccination remains as one of the challenges in public health. This study purposed to demonstrate the related factors of delayed diphtheria-tetanus-pertussis (DTP) vaccination using life table approach. A historical cohort study conducted in the poor areas of five large Iran cities. Totally, 3610 children with 24-47 months old age who had documented vaccination card were enrolled. Time of vaccination for the third dose of DTP vaccine was calculated. Life table survival was used to calculate the proportional probability of vaccination in each time. Wilcoxon test was used for the comparison proportional probability of delayed vaccination based on studies factors. The overall median delayed time for DTP3 was 38.52 days. The Wilcoxon test showed that city, nationality, education level of parents, birth order and being in rural areas are related to the high probability of delay time for DTP3 vaccination (P < 0. 001). Moreover, child gender and parent's job were not significant factors (P > 0.05). Being away from the capital, a high concentration of immigrants in the city borders with a low socioeconomic class leads to prolonged delay in DTP vaccination time. Special attention to these areas is needed to increase the levels of parental knowledge and to facilitate access to the health services care.

  16. A diphtheria toxin resistance marker for in vitro and in vivo selection of stably transduced human cells

    PubMed Central

    Picco, Gabriele; Petti, Consalvo; Trusolino, Livio; Bertotti, Andrea; Medico, Enzo

    2015-01-01

    We developed a selectable marker rendering human cells resistant to Diphtheria Toxin (DT). The marker (DTR) consists of a primary microRNA sequence engineered to downregulate the ubiquitous DPH2 gene, a key enzyme for the biosynthesis of the DT target diphthamide. DTR expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth. DTR-based selection efficiency and stability were comparable to those of established drug-resistance markers. As mice are insensitive to DT, DTR-based selection can be also applied in vivo. Direct injection of a GFP-DTR lentiviral vector into human cancer cell-line xenografts and patient-derived tumorgrafts implanted in mice, followed by systemic DT administration, yielded tumors entirely composed of permanently transduced cells and detectable by imaging systems. This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals. PMID:26420058

  17. Antibody persistence up to 5 y after vaccination with a quadrivalent meningococcal ACWY-tetanus toxoid conjugate vaccine in adolescents.

    PubMed

    Quiambao, Beatriz P; Bavdekar, Ashish; Dubey, Anand Prakash; Jain, Hemant; Kolhe, Devayani; Bianco, Véronique; Miller, Jacqueline M; Van der Wielen, Marie

    2017-02-02

    Long-term protection against meningococcal disease relies on antibody persistence after vaccination. We report antibody persistence up to 5 y after vaccination in adolescents who received a single dose of either meningococcal serogroups A, C, W, Y tetanus toxoid conjugate vaccine (MenACWY-TT, Pfizer) or MenACWY polysaccharide vaccine (MenPS, GSK Vaccines) at the age of 11-17 y in the randomized controlled primary study NCT00464815. In this phase III, open, controlled, multi-center persistence follow-up study conducted in India and the Philippines (NCT00974363), antibody persistence was evaluated by a serum bactericidal antibody assay using rabbit complement (rSBA) yearly, up to year 5 after vaccination. Serious adverse events (SAEs) related to study participation were recorded. Five years after a single dose of MenACWY-TT, the percentage of participants (N = 236) with rSBA titers ≥1:8 was 97.5% for serogroup A, 88.6% for serogroup C, 86.0% for serogroup W and 96.6% for serogroup Y. The percentages in the MenPS group (N = 86) were 93.0%, 87.1%, 34.9% and 66.3%, respectively. Exploratory analysis indicated a higher percentage of subjects with rSBA titers ≥1:8 for serogroups W and Y, and higher rSBA geometric mean antibody titers for serogroups A, W and Y in the MenACWY-TT group than the MenPS group at each time point (years 3, 4 and 5). No differences between groups were observed for serogroup C. No SAEs related to study participation were reported. In conclusion, the results of this follow-up study indicate that antibodies persisted up to 5 y after a single dose of MenACWY-TT in adolescents.

  18. The fully synthetic MAG-Tn3 therapeutic vaccine containing the tetanus toxoid-derived TT830-844 universal epitope provides anti-tumor immunity.

    PubMed

    Laubreton, Daphné; Bay, Sylvie; Sedlik, Christine; Artaud, Cécile; Ganneau, Christelle; Dériaud, Edith; Viel, Sophie; Puaux, Anne-Laure; Amigorena, Sebastian; Gérard, Catherine; Lo-Man, Richard; Leclerc, Claude

    2016-03-01

    Malignant transformations are often associated with aberrant glycosylation processes that lead to the expression of new carbohydrate antigens at the surface of tumor cells. Of these carbohydrate antigens, the Tn antigen is particularly highly expressed in many carcinomas, especially in breast carcinoma. We designed MAG-Tn3, a fully synthetic vaccine based on three consecutive Tn moieties that are O-linked to a CD4+ T cell epitope, to induce anti-Tn antibody responses that could be helpful for therapeutic vaccination against cancer. To ensure broad coverage within the human population, the tetanus toxoid-derived peptide TT830-844 was selected as a T-helper epitope because it can bind to various HLA-DRB molecules. We showed that the MAG-Tn3 vaccine, which was formulated with the GSK proprietary immunostimulant AS15 and designed for human cancer therapy, is able to induce an anti-Tn antibody response in mice of various H-2 haplotypes, and this response correlates with the ability to induce a specific T cell response against the TT830-844 peptide. The universality of the TT830-844 peptide was extended to new H-2 and HLA-DRB molecules that were capable of binding this T cell epitope. Finally, the MAG-Tn3 vaccine was able to induce anti-Tn antibody responses in cynomolgus monkeys, which targeted Tn-expressing tumor cells and mediated tumor cell death both in vitro and in vivo. Thus, MAG-Tn3 is a highly promising anticancer vaccine that is currently under evaluation in a phase I clinical trial.

  19. Antibody persistence up to 5 y after vaccination with a quadrivalent meningococcal ACWY-tetanus toxoid conjugate vaccine in adolescents

    PubMed Central

    Quiambao, Beatriz P.; Bavdekar, Ashish; Dubey, Anand Prakash; Jain, Hemant; Kolhe, Devayani; Bianco, Véronique; Miller, Jacqueline M.; Van der Wielen, Marie

    2017-01-01

    ABSTRACT Long-term protection against meningococcal disease relies on antibody persistence after vaccination. We report antibody persistence up to 5 y after vaccination in adolescents who received a single dose of either meningococcal serogroups A, C, W, Y tetanus toxoid conjugate vaccine (MenACWY-TT, Pfizer) or MenACWY polysaccharide vaccine (MenPS, GSK Vaccines) at the age of 11–17 y in the randomized controlled primary study NCT00464815. In this phase III, open, controlled, multi-center persistence follow-up study conducted in India and the Philippines (NCT00974363), antibody persistence was evaluated by a serum bactericidal antibody assay using rabbit complement (rSBA) yearly, up to year 5 after vaccination. Serious adverse events (SAEs) related to study participation were recorded. Five years after a single dose of MenACWY-TT, the percentage of participants (N = 236) with rSBA titers ≥1:8 was 97.5% for serogroup A, 88.6% for serogroup C, 86.0% for serogroup W and 96.6% for serogroup Y. The percentages in the MenPS group (N = 86) were 93.0%, 87.1%, 34.9% and 66.3%, respectively. Exploratory analysis indicated a higher percentage of subjects with rSBA titers ≥1:8 for serogroups W and Y, and higher rSBA geometric mean antibody titers for serogroups A, W and Y in the MenACWY-TT group than the MenPS group at each time point (years 3, 4 and 5). No differences between groups were observed for serogroup C. No SAEs related to study participation were reported. In conclusion, the results of this follow-up study indicate that antibodies persisted up to 5 y after a single dose of MenACWY-TT in adolescents. PMID:28152332

  20. Vibrio cholerae O139 Conjugate Vaccines: Synthesis and Immunogenicity of V. cholerae O139 Capsular Polysaccharide Conjugates with Recombinant Diphtheria Toxin Mutant in Mice

    PubMed Central

    Kossaczka, Zuzana; Shiloach, Joseph; Johnson, Virginia; Taylor, David N.; Finkelstein, Richard A.; Robbins, John B.; Szu, Shousun C.

    2000-01-01

    Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide of Vibrio cholerae O1 (lipopolysaccharide) and of Vibrio cholerae O139 (capsular polysaccharide [CPS]) is associated with immunity to the homologous pathogen. The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates). Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates of V. cholerae O139 CPS with the recombinant diphtheria toxin mutant, CRMH21G. Adipic acid dihydrazide was used as a linker. When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulated V. cholerae O139. Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity. These results indicate that the conjugates elicited IgG with vibriocidal activity. Conjugates also elicited high levels of serum diphtheria toxin IgG. Convalescent sera from 20 cholera patients infected with V. cholerae O139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to ≤50. Treatment with 2-ME reduced the titers of 17 of 20 patients to ≤50. These data show that, like infection with V. cholerae O1, infection with V. cholerae O139 induces vibriocidal antibodies specific to the surface polysaccharide of this bacterium (CPS) that are mostly of IgM class. Based on these data, clinical trials with the V. cholerae O139 CPS conjugates with recombinant diphtheria toxin are planned. PMID:10948122

  1. A slow-forming isopeptide bond in the structure of the major pilin SpaD from Corynebacterium diphtheriae has implications for pilus assembly.

    PubMed

    Kang, Hae Joo; Paterson, Neil G; Kim, Chae Un; Middleditch, Martin; Chang, Chungyu; Ton-That, Hung; Baker, Edward N

    2014-05-01

    The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed by C. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys-Asn isopeptide bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to bond formation. Two crystal structures, of the full-length three-domain protein at 2.5 Å resolution and of a two-domain (D2-D3) construct at 1.87 Å resolution, show that each of the three Ig-like domains contains a single Lys-Asn isopeptide-bond cross-link, assumed to give mechanical stability as in other such pili. Additional stabilizing features include a disulfide bond in the D3 domain and a calcium-binding loop in D2. The N-terminal D1 domain is more flexible than the others and, by analogy with other major pilins of this type, the slow formation of its isopeptide bond can be attributed to its location adjacent to the lysine used in sortase-mediated polymerization during pilus assembly.

  2. Durability of immunity to diphtheria, tetanus and poliomyelitis after a three dose immunization schedule completed in the first eight months of life.

    PubMed

    Jones, A E; Johns, A; Magrath, D I; Melville-Smith, M; Sheffield, F

    1989-08-01

    Blood samples were obtained from school entrants whose primary immunization schedule had consisted of three doses of DT or DTP vaccine and three doses of OPV all given before the age of 8 months. The sera were separated and assayed for diphtheria antitoxin, tetanus antitoxin and antibodies to the three serotypes of poliovirus. The results of the assays showed that the abbreviated three dose schedule induced satisfactory immunity to all five infections until school entry and that a reinforcing dose at 18 months was unnecessary.

  3. Immunogenicity and reactogenicity of two diphtheria-tetanus-whole cell pertussis vaccines in Iranian pre-school children, a randomized controlled trial.

    PubMed

    Zarei, Saeed; Jeddi-Tehrani, Mahmood; Mehdi Akhondi, Mohammad; Zeraati, Hojjat; Ferydonfar, Amir Ali; Nasernia, Jalaledin; Tavangar, Banafsheh; Shokri, Fazel

    2013-06-01

    The present study was undertaken to compare the immunogenicity and reactogenicity of two diphtheria-tetanus-whole cell pertussis (DTwP) vaccines administered to Iranian preschool children. In this randomized, double-blind and multicenter prospective study, 672 children aged 4-6 y were administered with either a local DTwP vaccine (DTwP-Local) (n = 337) or a commercial vaccine (DTwP-Pasteur) (n = 335). All subjects received DTwP vaccine at 4-6 y of age, following the national immunization schedule of Iran. Blood samples were collected before and 2-4 weeks after the vaccination. Immunogenicity of each vaccine was assessed by ELISA using commercial kits. Reactogenicity was assessed by the parents for seven days post-booster using diary cards. The geometric mean titers (GMTs) of the antibodies induced against diphtheria and tetanus by DTwP-Local were 7.7 and 9.4 IU/ml and those of DTwP-Pasteur were 8.2 and 8.6 IU/ml, respectively. There was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. The GMTs of antibodies produced against pertussis were 30.2 EU/ml for DTwP-Local and 47.9 EU/ml for DTwP-Pasteur vaccines (p<0.001). Pain and fever (axillary temperature>37.5°C) were the most frequent local and systemic reactions observed after the vaccination. All local and systemic reactions observed after vaccination were significantly higher in subjects immunized with DTwP-Local vaccine. Immunogenicity against diphtheria and tetanus was similar for the two vaccines, but immunogenicity of the local vaccine against pertussis was significantly less efficient than that of DTwP-Pasteur. This difference and the higher side effects of the DTwP-Local vaccine could be due to the bacterial strain or the preparation or formulation protocol of the local pertussis vaccine.

  4. Effect of Tetanus-diphtheria Vaccine on Immune Response to Hepatitis B Vaccine in Low-responder Individuals

    PubMed Central

    Haghighat, Abbas; Moafi, Mohammad; Sharifian, Jalil; Salehi, Hassan; Taleban, Roya; Kalbasi, Nader; Salehi, Marzieh; Salehi, Mohammad Mahdi; Salehi, Maryam

    2016-01-01

    Background: Conventional hepatitis B virus (HBV) vaccination fails to achieve efficient protection in about 5–10% of the world population. Hence, different strategies have been adopted to ameliorate HBV antibody titers. This study aimed to evaluate the concurrent application of tetanus-diphtheria (Td) and HBV vaccination on hepatitis B surface (HBs) antibody titer in low-responder healthy individuals. Methods: This was a randomized clinical trial, which was implemented among 140 of medical staff working as health-care workers assumed as low-responders. The subjects were randomly allocated to either control or interventional groups. The control and interventional groups received HBV recombinant vaccine while the latter group was also vaccinated through Td. Enzyme-linked immunosorbent assay was applied to measure HBs antibody (HBsAb) titers just before and 6 months after the last vaccination. All data were entered into SPSS software. Independent t-test, paired t-test, and Chi-square or Fisher's exact test were applied for data comparison. Results: Antibody titers of the subjects in the intervention and control groups soared from 49.08 ± 20.08 IU/L to 917.78 ± 204.80 IU/L and from 46.95 ± 18.55 to 586.81 ± 351.77 IU/L, respectively (both P < 0.001); nevertheless, by comparison with control group, variation of antibody titer in the interventional group was significantly higher (P < 0.001). Conclusions: Concurrent application of Td and HBV vaccine could effectively enhance protective levels of HBsAb titers in low-responder individuals. PMID:27563430

  5. Hydrogen–Deuterium Exchange and Mass Spectrometry Reveal the pH-Dependent Conformational Changes of Diphtheria Toxin T Domain

    PubMed Central

    2015-01-01

    The translocation (T) domain of diphtheria toxin plays a critical role in moving the catalytic domain across the endosomal membrane. Translocation/insertion is triggered by a decrease in pH in the endosome where conformational changes of T domain occur through several kinetic intermediates to yield a final trans-membrane form. High-resolution structural studies are only applicable to the static T-domain structure at physiological pH, and studies of the T-domain translocation pathway are hindered by the simultaneous presence of multiple conformations. Here, we report the application of hydrogen–deuterium exchange mass spectrometry (HDX-MS) for the study of the pH-dependent conformational changes of the T domain in solution. Effects of pH on intrinsic HDX rates were deconvolved by converting the on-exchange times at low pH into times under our “standard condition” (pH 7.5). pH-Dependent HDX kinetic analysis of T domain clearly reveals the conformational transition from the native state (W-state) to a membrane-competent state (W+-state). The initial transition occurs at pH 6 and includes the destabilization of N-terminal helices accompanied by the separation between N- and C-terminal segments. The structural rearrangements accompanying the formation of the membrane-competent state expose a hydrophobic hairpin (TH8–9) to solvent, prepare it to insert into the membrane. At pH 5.5, the transition is complete, and the protein further unfolds, resulting in the exposure of its C-terminal hydrophobic TH8–9, leading to subsequent aggregation in the absence of membranes. This solution-based study complements high resolution crystal structures and provides a detailed understanding of the pH-dependent structural rearrangement and acid-induced oligomerization of T domain. PMID:25290210

  6. Cell-specific modulation of drug resistance in acute myeloid leukemic blasts by diphtheria fusion toxin, DT388-GMCSF.

    PubMed

    Frankel, A E; Hall, P D; McLain, C; Safa, A R; Tagge, E P; Kreitman, R J

    1998-01-01

    Radiochemotherapy-resistant blasts commonly cause treatment failure in acute myeloid leukemia (AML), and their resistance is due, in part, to overexpression of multidrug resistance (mdr) proteins. We reasoned that targeted delivery of protein synthesis inactivating toxins to leukemic blasts would reduce the cellular concentrations of relatively short half-life resistance proteins and sensitize the cells to cytotoxic drugs. To test this hypothesis, we employed human granulocyte-macrophage colony-stimulating factor fused to truncated diphtheria toxin (DT388-GMCSF). The human AML cell line HL60 and its vincristine-resistant sublines, HL60Vinc and HL60VCR, were incubated in vitro for 24 h with varying concentrations of toxin. Doxorubicin was added for an additional 24 h, and cell cytotoxicity was assayed by thymidine incorporation and colony formation in semisolid medium. DT388-GMCSF sensitized HL60Vinc and HL60VCR but not HL60 to doxorubicin. Combination indices for three log cell kill varied from 0.2 to 0.3. In contrast, pretreatment with doxorubicin followed by toxins failed to show synergy. At least in the case of the vincristine-resistant cell lines, modulation of drug resistance correlated with reduction in membrane P-glycoprotein concentrations based on immunoblots with C219 antibody, flow cytometry with MRK16 antibody, and cell uptake of doxorubicin. These observations suggest clinical trials of combination therapy may be warranted in patients with refractory AML. Further, targeted toxins may represent a novel class of cell-specific modulators of drug resistance for a number of malignancies.

  7. Targeting glioblastoma multiforme with an IL-13/diphtheria toxin fusion protein in vitro and in vivo in nude mice.

    PubMed

    Li, Chunbin; Hall, Walter A; Jin, Ni; Todhunter, Deborah A; Panoskaltsis-Mortari, Angela; Vallera, Daniel A

    2002-05-01

    Fusion proteins composed of tumor binding agents and potent catalytic toxins show promise for intracranial therapy of brain cancer and an advantage over systemic therapy. Glioblastoma multiforme (GBM) is the most common form of brain cancer and overexpresses IL-13R. Thus, we developed an interleukin-13 receptor targeting fusion protein, DT(390)IL13, composed of human interleukin-13 and the first 389 amino acids of diphtheria toxin. To measure its ability to inhibit GBM, DT(390)IL13 was tested in vitro and found to inhibit selectively the U373 MG GBM cell line with an IC(50) around 12 pmol/l. Cytotoxicity was neutralized by anti-human-interleukin-13 antibody, but not by control antibodies. In vivo, small U373 MG glioblastoma xenografts in nude mice completely regressed in most animals after five intratumoral injections of 1 microg of DT(390)IL13 q.o.d., but not by the control fusion protein DT(390)IL-2. DT(390)IL13 was also tested against primary explant GBM cells of a patient's excised tumor and the IC(50) was similar to that measured for U373 MG. Further studies showed a therapeutic window for DT(390)IL13 of 1-30 microg/injection and histology studies and enzyme measurements showed that the maximum tolerated dose of DT(390)IL13 had little effect on kidney, liver, spleen, lung and heart in non-tumor-bearing immunocompetent mice. Together, these data suggest that DT(390)IL13 may provide an important, alternative therapy for brain cancer.

  8. Universal tetanus, diphtheria, acellular pertussis (Tdap) vaccination of adults: What Canadian health care providers know and need to know

    PubMed Central

    MacDougall, D; Halperin, BA; MacKinnon-Cameron, D; Li, L; McNeil, SA; Langley, JM; Halperin, SA

    2015-01-01

    The tetanus, diphtheria, and acellular pertussis vaccine (Tdap) is recommended for all adults in both Canada and the United States. There are few data on the proportion of Canadian adults vaccinated with Tdap; however, anecdotal reports indicate that uptake is low. This study aimed to explore the knowledge, attitudes, beliefs, and behaviors of Canadian health care providers (HCPs) in an attempt to identify potential barriers and facilitators to Tdap uptake. HCPs were surveyed and a geographic and practice representative sample was obtained (N =1,167). In addition, 8 focus groups and 4 interviews were conducted nationwide. Results from the survey indicate that less than half (47.5%) of all respondents reported being immunized with Tdap themselves, while 58.5% routinely offer Tdap to their adult patients. Knowledge scores were relatively low (63.2% correct answers). The best predictor of following the adult Tdap immunization guidelines was awareness of and agreement with those recommendations. Respondents who were aware of the recommendations were more likely to think that Tdap is safe and effective, that their patients are at significant risk of getting pertussis, and to feel that they have sufficient information (p < 0.0001 for each statement). Focus group data supported the survey results and indicated that there are substantial gaps in knowledge of pertussis and Tdap among Canadian HCPs. Lack of public knowledge about adult immunization, lack of immunization registries, a costing differential between Td and Tdap, workload required to deliver the vaccine, and vaccine hesitancy were identified as barriers to compliance with the national recommendations for universal adult immunization, and suggestions were provided to better translate recommendations to front-line practitioners. PMID:26090861

  9. Anti-human HB-EGF monoclonal antibodies inhibiting ectodomain shedding of HB-EGF and diphtheria toxin binding.

    PubMed

    Hamaoka, Miki; Chinen, Ichino; Murata, Takuya; Takashima, Seiji; Iwamoto, Ryo; Mekada, Eisuke

    2010-07-01

    HB-EGF is a member of the EGF family of growth factors that bind and activate the EGF receptor. HB-EGF is synthesized as a membrane-anchored protein (proHB-EGF), and then proteolytically cleaved, resulting in the mitogenically active soluble form. ProHB-EGF functions as the receptor for the diphtheria toxin (DT). HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Monoclonal antibodies (mAbs) specific for HB-EGF could be an important tool in HB-EGF research. However, few such mAbs have been established to date. In this study, we newly generated seven clones of hybridoma-derived mAbs by immunizing HB-EGF null mice with recombinant human HB-EGF protein. All mAbs specifically bound to human HB-EGF but not to mouse HB-EGF. Epitope mapping analysis showed that most of the mAbs recognized the EGF-like domain. Although none of the newly isolated mAbs directly inhibited the mitogenic activity of HB-EGF for EGFR-expressing cells, some strongly inhibited DT-binding. Interestingly, some of the mAbs efficiently inhibited ectodomain shedding of proHB-EGF, and consequently prevented the cell growth of the EGFR-expressing cells in a co-culture system with proHB-EGF-expressing cells. Hence, these new anti-HB-EGF mAbs may advance clinical as well as basic research on HB-EGF.

  10. Mixing of diphtheria, tetanus, and acellular pertussis (DTaP) vaccines in a population of children in managed care

    PubMed Central

    Masseria, Cristina; Buikema, Ami R; Liu, Fang; Krishnarajah, Girishanthy

    2015-01-01

    The Advisory Committee on Immunization Practices recommends administering diphtheria, tetanus and acellular pertussis (DTaP) vaccines to children at 2, 4, 6, 15–18 months, and 4–6 y of age; preferably with the same-brand vaccine for the whole series. We estimated age-appropriate DTaP dose completion and the proportion of children receiving a “mixed” DTaP vaccination series (ie, including DTaP vaccines from ≥2 brands) across the 3 milestones. Commercially-insured children born between 01/01/2003 and 04/30/2011 were identified from United States health insurance claims data and assigned to ≥1 of 3 study cohorts based on the duration of continuous health plan enrollment: 1) birth to <8 months; 2) birth to <20 months; 3) birth to <7 years. Dose completion and brand mixing of the first 3, first 4 or all 5 doses were measured in the respective cohorts. Administered DTaP vaccinations were identified in claims data and classified by brand (based on vaccine components and manufacturer). The analysis included children who received ≥2 DTaP vaccinations and had known brand information for all doses. Age-appropriate dose completion was 77% with 3 doses (<8 months cohort), 71% with 4 doses (<20 months cohort), and 85% with 5 doses (<7 years cohort). Mixed DTaP series were received by 4.7% (95% confidence interval [CI]: 4.6%-4.7%) in the <8 months cohort, 29.0% (95% CI: 28.6%–29.4%) in the <20 months cohort, and 39.0% (95% CI: 34.5, 43.6) in the <7 years cohort. DTaP mixing was just 4.7% for the first 3 doses but subsequently increased with the number of administered doses. PMID:25714800

  11. Treatment of ovarian cancer ascites by intra-peritoneal injection of diphtheria toxin A chain-H19 vector: a case report

    PubMed Central

    2010-01-01

    Introduction Ovarian cancer ascitic fluid, which contains malignant cells, is usually present in women with an advanced stage disease. There are currently no effective therapies for the treatment of ovarian cancer ascitic fluid. We developed a new therapeutic strategy to target expression of the diphtheria toxin fragment A gene in ovarian tumor cells under the control of H19 regulatory sequences. Case presentation A 64-year-old Caucasian woman was diagnosed with a stage IIIc epithelial ovarian cancer. She suffered from progressive disease, accumulation of malignant ascites that needed to be drained weekly, abdominal pain, vomiting, anorexia and severe weakness. Infusion of the diphtheria toxin A chain-H19 plasmid into the peritoneum of our patient resulted in complete resolution of the ascites with minimum adverse events. Conclusion On the basis of this preliminary experience, we are currently conducting an extensive Phase I study on a larger number of patients in order to assess the safety and preliminary efficacy of this novel patient-oriented treatment approach. PMID:20663201

  12. Crystal Structure of DsbA from Corynebacterium diphtheriae and Its Functional Implications for CueP in Gram-Positive Bacteria.

    PubMed

    Um, Si-Hyeon; Kim, Jin-Sik; Song, Saemee; Kim, Nam Ah; Jeong, Seong Hoon; Ha, Nam-Chul

    2015-08-01

    In Gram-negative bacteria in the periplasmic space, the dimeric thioredoxin-fold protein DsbC isomerizes and reduces incorrect disulfide bonds of unfolded proteins, while the monomeric thioredoxin-fold protein DsbA introduces disulfide bonds in folding proteins. In the Gram-negative bacteria Salmonella enterica serovar Typhimurium, the reduced form of CueP scavenges the production of hydroxyl radicals in the copper-mediated Fenton reaction, and DsbC is responsible for keeping CueP in the reduced, active form. Some DsbA proteins fulfill the functions of DsbCs, which are not present in Gram-positive bacteria. In this study, we identified a DsbA homologous protein (CdDsbA) in the Corynebacterium diphtheriae genome and determined its crystal structure in the reduced condition at 1.5 Å resolution. CdDsbA consists of a monomeric thioredoxin-like fold with an inserted helical domain and unique N-terminal extended region. We confirmed that CdDsbA has disulfide bond isomerase/reductase activity, and we present evidence that the N-terminal extended region is not required for this activity and folding of the core DsbA-like domain. Furthermore, we found that CdDsbA could reduce CueP from C. diphtheriae.

  13. Four is better than nine. a combined diphtheria-tetanus-pertussis-hepatitis B-Haemophilus influenzae type b vaccine for routine immunization in Malaysia.

    PubMed

    Wong, Swee-Lan; Soosai, Padma; Teoh, Yee-Leong; Han, Htay-Htay; Lefevre, Inge; Bock, Hans L

    2008-05-01

    Malaysian infants would have to receive nine injections during the first few months of life in order to be protected against disease caused by hepatitis B (HBV), diphtheria, tetanus, pertussis and Haemophilus influenzae type b (Hib) if single HBV and Hib vaccines were used. We evaluated a combined DTPw-HBV/Hib vaccine administered at 1.5, 3 and 5 months after a birth dose of hepatitis B vaccine (HBV). One month after completion of the primary vaccination, 99% of subjects had seroprotective anti-HBV antibody levels, and at least 98% had seroprotective antibodies against diphtheria, tetanus, and Hib, and were seropositive for pertussis antibodies. The immune response to the combined vaccine was comparable to that induced by separate injections with DTPw, HBV and Hib vaccines. Overall, the DTPw-HBV/Hib vaccine was as well tolerated as separate administration of DTPw, HBV and Hib vaccines. The combined DTPw-HBV/Hib vaccine induces protection against five diseases as recommended in the Malaysian routine vaccination schedule. Use of the combined DTPw-HBV/Hib vaccine can reduce the required number of injections from nine to four in the first few months of life.

  14. The safety and reactogenicity of a reduced-antigen-content diphtheria-tetanus-acellular pertussis (dTpa) booster vaccine in healthy Vietnamese children.

    PubMed

    Anh, Dang Duc; Jayadeva, Girish; Kuriyakose, Sherine; Han, Htay Htay

    2016-08-17

    Despite effective infant immunization against pertussis, the disease continues to circulate due to waning immunity. Booster vaccinations against pertussis beyond infancy are widely recommended. In Vietnam, however, no recommendations for pertussis boosters beyond the second year of life exist. This open-label, single-centre study was designed to assess the safety of a single booster dose of reduced-antigen-content-diphtheria-tetanus-acellular-pertussis vaccine (dTpa) in 300 healthy Vietnamese children (mean age 7.9years), who had completed primary vaccination against diphtheria, tetanus and pertussis. Solicited symptoms were recorded for 4days and unsolicited and serious adverse events (SAEs) for 31days post-vaccination. Pain and fatigue were the most common solicited local and general symptoms in 35.0% and 14.0% of children, respectively. Grade 3 swelling occurred in 3 children; no large injection site reactions or SAEs were reported. The dTpa booster vaccine was well tolerated and this study supports its administration in school age Vietnamese children.

  15. Use of a Novel Integrase-Deficient Lentivirus for Targeted Anti-Cancer Therapy With Survivin Promoter-Driven Diphtheria Toxin A

    PubMed Central

    Lin, Baoshun; Gao, Anding; Zhang, Rui; Ma, Hongyu; Shen, Haifeng; Hu, Qiong; Zhang, Hua; Zhao, Meng; Lan, Xiaopeng; Liu, Kuancan

    2015-01-01

    Abstract As an immunotoxin, diphtheria toxin has been widely used in gene therapy and gene function assays for its roles in protein synthesis inhibition, and the aim of our study is to set up a nonintegrating lentiviral system for specific expression of diphtheria toxin A (DTA) used in cancer gene therapy. Here, we established a lentiviral system that could coordinately express fluorescent protein and DTA driven by the cytomegalovirus (CMV) promoter, which is convenient for us to precisely trace the expression of DTA and monitor the process of lentivirus packaging. To achieve safer cancer therapy, we replaced the CMV promoter with the Survivin promoter, a specific promoter that is dramatically activated in cancer tissues and cells, but not in normal tissues and cells, and that will impose greater therapeutic potential because a significant expression difference occurred between these 2 groups. Meanwhile, we obtained integrase-deficient lentivirus (IDLV) after packaging with the integrase mutant, which expresses defective integrase RRK262263264AAH, to minimize the side effects that derived from the insertional mutagenesis of the host genome. Our results suggest that the IDLV system that we generated possesses therapeutic potential in cancers in vitro and in vivo. PMID:26252309

  16. Schistosoma mansoni Infection Can Jeopardize the Duration of Protective Levels of Antibody Responses to Immunizations against Hepatitis B and Tetanus Toxoid

    PubMed Central

    Riner, Diana K.; Ndombi, Eric M.; Carter, Jennifer M.; Omondi, Amos; Kittur, Nupur; Kavere, Emmy; Korir, Harrison K.; Flaherty, Briana; Karanja, Diana; Colley, Daniel G.

    2016-01-01

    Background Schistosomiasis is a disease of major public health importance in sub-Saharan Africa. Immunoregulation begins early in schistosome infection and is characterized by hyporesponsiveness to parasite and bystander antigens, suggesting that a schistosome infection at the time of immunization could negatively impact the induction of protective vaccine responses. This study examined whether having a Schistosoma mansoni infection at the time of immunization with hepatitis B and tetanus toxoid (TT) vaccines impacts an individual’s ability to achieve and maintain protective antibody levels against hepatitis B surface antigen or TT. Methods Adults were recruited from Kisumu Polytechnic College in Western Kenya. At enrollment, participants were screened for schistosomiasis and soil transmitted helminths (STHs) and assigned to groups based on helminth status. The vaccines were then administered and helminth infections treated a week after the first hepatitis B boost. Over an 8 month period, 3 blood specimens were obtained for the evaluation of humoral and cytokine responses to the vaccine antigens and for immunophenotyping. Results 146 individuals were available for final analysis and 26% were S. mansoni positive (Sm+). Schistosomiasis did not impede the generation of initial minimum protective antibody levels to either hepatitis B or TT vaccines. However, median hepatitis B surface antibody levels were significantly lower in the Sm+ group after the first boost and remained lower, but not significantly lower, following praziquantel (PZQ) treatment and final boost. In addition, 8 months following TT boost and 7 months following PZQ treatment, Sm+ individuals were more likely to have anti-TT antibody levels fall below levels considered optimal for long term protection. IL-5 levels in response to in vitro TT stimulation of whole blood were significantly higher in the Sm+ group at the 8 month time period as well. Conclusions Individuals with schistosomiasis at the

  17. Efficacy, but not antibody titer or affinity, of a heroin hapten conjugate vaccine correlates with increasing hapten densities on tetanus toxoid, but not on CRM197 carriers.

    PubMed

    Jalah, Rashmi; Torres, Oscar B; Mayorov, Alexander V; Li, Fuying; Antoline, Joshua F G; Jacobson, Arthur E; Rice, Kenner C; Deschamps, Jeffrey R; Beck, Zoltan; Alving, Carl R; Matyas, Gary R

    2015-06-17

    Vaccines against drugs of abuse have induced antibodies in animals that blocked the biological effects of the drug by sequestering the drug in the blood and preventing it from crossing the blood-brain barrier. Drugs of abuse are too small to induce antibodies and, therefore, require conjugation of drug hapten analogs to a carrier protein. The efficacy of these conjugate vaccines depends on several factors including hapten design, coupling strategy, hapten density, carrier protein selection, and vaccine adjuvant. Previously, we have shown that 1 (MorHap), a heroin/morphine hapten, conjugated to tetanus toxoid (TT) and mixed with liposomes containing monophosphoryl lipid A [L(MPLA)] as adjuvant, partially blocked the antinociceptive effects of heroin in mice. Herein, we extended those findings, demonstrating greatly improved vaccine induced antinociceptive effects up to 3% mean maximal potential effect (%MPE). This was obtained by evaluating the effects of vaccine efficacy of hapten 1 vaccine conjugates with varying hapten densities using two different commonly used carrier proteins, TT and cross-reactive material 197 (CRM197). Immunization of mice with these conjugates mixed with L(MPLA) induced very high anti-1 IgG peak levels of 400-1500 μg/mL that bound to both heroin and its metabolites, 6-acetylmorphine and morphine. Except for the lowest hapten density for each carrier, the antibody titers and affinity were independent of hapten density. The TT carrier based vaccines induced long-lived inhibition of heroin-induced antinociception that correlated with increasing hapten density. The best formulation contained TT with the highest hapten density of ≥30 haptens/TT molecule and induced %MPE of approximately 3% after heroin challenge. In contrast, the best formulation using CRM197 was with intermediate 1 densities (10-15 haptens/CRM197 molecule), but the %MPE was approximately 13%. In addition, the chemical synthesis of 1, the optimization of the conjugation

  18. Efficacy and safety of vi-tetanus toxoid conjugated typhoid vaccine (PedaTyph™) in Indian children: School based cluster randomized study

    PubMed Central

    Mitra, Monjori; Shah, Nitin; Ghosh, Apurba; Chatterjee, Suparna; Kaur, Iqbal; Bhattacharya, Nisha; Basu, Suparna

    2016-01-01

    ABSTRACT Vi polysaccharide typhoid vaccines cannot be used in children <2 years owing to poor immunogenic and T cell independent properties. Conjugate vaccine prepared by binding Vi to tetanus toxoids (Vi-TT) induces protective levels even in children <2 years. We evaluated efficacy and safety following vaccination with a Vi-TT vaccine in children 6 months to 12 years of age. Overall, 1765 subjects were recruited from two registered municipal urban slums of southern Kolkata. Most of the children of the slum dwellers attended the schools in the locality which was selected with permission from the school authority. Schools were randomly divided into vaccinated (Test group) and unvaccinated group (Control group). Children and their siblings of test group received 2-doses of PedaTyph™ vaccine at 6 weeks interval. Control group received vaccines as per national guidelines. Adverse events (AEs) were examined after 30 minutes, 1 month and clinical events were observed till 12 months post-vaccination. Incidence of culture positive typhoid fever in the control group was 1.27% vis-a-vis none in vaccine group during 12 months. In subgroup evaluated for immunogenicity, an antibody titer value of 1.8 EU/ml (95% CI: 1.5 EU/ml, 2.2 EU/ml), 32 EU/ml (95% CI: 27.0 EU/ml, 39.0 EU/ml) and 14 EU/ml (95% CI: 12.0 EU/ml, 17.0 EU/ml) at baseline, 6 weeks and 12 months, respectively was observed. Sero-conversion among the sub-group was 100% after 6 weeks of post-vaccination and 83% after 12 months considering 4-fold rise from baseline. The efficacy of vaccine was 100 % (95% CI: 97.6%, 100%) in the first year of follow-up with minimal AEs post vaccination. Vi conjugate typhoid vaccine conferred 100% protection against typhoid fever in 1765 children 6 months to 12 years of age with high immunogenicity in a subgroup from the vaccine arm. PMID:26901576

  19. Human immunoglobulin repertoires against tetanus toxoid contain a large and diverse fraction of high-affinity promiscuous V(H) genes.

    PubMed

    de Kruif, John; Kramer, Arjen; Visser, Therèse; Clements, Carina; Nijhuis, Roy; Cox, Freek; van der Zande, Vanessa; Smit, Renate; Pinto, Daniel; Throsby, Mark; Logtenberg, Ton

    2009-04-03

    To study the contribution of antibody light (L) chains to the diversity and binding properties of immune repertoires, a phage display repertoire was constructed from a single human antibody L chain and a large collection of antibody heavy (H) chains harvested from the blood of two human donors immunized with tetanus toxoid (TT) vaccine. After selection for binding to TT, 129 unique antibodies representing 53 variable immunoglobulin H chain (V(H)) gene rearrangements were isolated. This panel of anti-TT antibodies restricted to a single variable immunoglobulin L chain (V(L)) could be organized into 17 groups binding non-competing epitopes on the TT molecule. Comparison of the V(H) regions in this V(L)-restricted panel with a previously published repertoire of anti-TT V(H) regions with cognate V(H)-V(L) pairing showed a very similar distribution of V(H), D(H) and J(H) gene segment utilization and length of the complementarity-determining region 3 of the H chain. Surface plasmon resonance analysis of the single-V(L) anti-TT repertoire unveiled a range of affinities, with a median monovalent affinity of 2 nM. When the single-V(L) anti-TT V(H) repertoire was combined with a collection of naïve V(L) regions and again selected for binding to TT, many of the V(H) genes were recovered in combination with a diversity of V(L) regions. The affinities of a panel of antibodies consisting of a single promiscuous anti-TT V(H) combined with 15 diverse V(L) chains were determined and found to be identical to each other and to the original isolate restricted to a single-V(L) chain. Based on previous estimates of the clonal size of the human anti-TT repertoire, we conclude that up to 25% of human anti-TT-encoding V(H) regions from an immunized repertoire have promiscuous features. These V(H) regions readily combine with a single antibody L chain to result in a large panel of anti-TT antibodies that conserve the expected epitope diversity, V(H) region diversity and affinity of a

  20. Inclusion of a universal tetanus toxoid CD4+ T cell epitope P2 significantly enhanced the immunogenicity of recombinant rotavirus ΔVP8* subunit parenteral vaccines

    PubMed Central

    Wen, Xiaobo; Wen, Ke; Cao, Dianjun; Li, Guohua; Jones, Ronald W.; Li, Jianping; Szu, Shousun; Hoshino, Yasutaka; Yuan, Lijuan

    2014-01-01

    Currently available live oral rotavirus vaccines, Rotarix® and RotaTeq®, are highly efficacious in developed countries. However, the immunogenicity and efficacy of such vaccines in some developing countries are low. We reported previously that bacterially-expressed rotavirus ΔVP8* subunit vaccine candidates with P[8], P[4] or P[6] specificity elicited high-titer virus neutralizing antibodies in animals immunized intramuscularly. Of note was the finding that antibodies induced with the P[8]ΔVP8* vaccine neutralized both homotypic P[8] and heterotypic P[4] rotavirus strains to high titer. To further improve its vaccine potential, a tetanus toxoid universal CD4+ T cell epitope P2 was introduced into P[8] or P[6]ΔVP8* construct. The resulting recombinant fusion proteins expressed in Escherichia coli were of high solubility and were produced with high yield. Two doses (10 or 20μg/dose) of the P2-P[8]ΔVP8* vaccine or P2-P[6]ΔVP8* vaccine with aluminum phosphate adjuvant elicited significantly higher geometric mean homologous neutralizing antibody titers than the vaccines without P2 in intramuscularly immunized guinea pigs. Interestingly, high levels of neutralizing antibody responses induced in guinea pigs with 3 doses of the P2-P[8]ΔVP8* vaccine persisted for at least 6 months. Furthermore, in the gnotobiotic piglet challenge study, three intramuscular doses (50μg/dose) of the P2-P[8]ΔVP8* vaccine with aluminum phosphate adjuvant significantly delayed the onset of diarrhea and significantly reduced the duration of diarrhea and the cumulative diarrhea score after oral challenge with virulent human rotavirus Wa (G1P[8]) strain. The P2-P[8]ΔVP8* vaccine induced serum virus neutralizing antibody and VP4-specific IgG antibody production prechallenge, and primed the pigs for higher antibody and intestinal and systemic virus-specific IFN-γ producing CD4+ T cell responses postchallenge. These two subunit vaccines could be used at a minimum singly or preferably in

  1. IMMUNOGENICITY AND SAFETY OF QUINVAXEM® (DIPHTHERIA, TETANUS, WHOLE-CELL PERTUSSIS, HEPATITIS B AND HAEMOPHILUS INFLUENZAE TYPE B VACCINE) GIVEN TO VIETNAMESE INFANTS AT 2 TO 4 MONTHS OF AGE.

    PubMed

    Huu, Tran Ngoc; Phuong, Nguyen Thi Minh; Toan, Nguyen Trong; Thang, Ho Vinh

    2015-07-01

    Vietnam plans to replace the routine childhood diphtheria, pertussis and tetanus combination (DPT) vaccine with a pentavalent vaccine. The present study was performed to assess the immunogenicity and safety of the combined diphtheria, tetanus, whole-cell pertussis, hepatitis B (HepB), and Haemophilus influenzae type b (Hib) (DTwP-HepB-Hib) Quinvaxem® vaccine in children. A total of 131 infants received the Quinvaxem® vaccine at 2, 3 and 4 months. Antibody levels were measured at baseline, at one month after the third injection and one year after the first injection. Seroprotection rates were high for each vaccine antigen at one month after the third dose: 93.1% for diphtheria, 98.5% for tetanus, 99.2% for pertussis (seroconversion rate), 93.1% for HepB, and 100% for Hib (anti-PRP ≥ 0.15 µg/ml). The rate of children with protective antibodies persisting at one year after the first dose was 88.4% for diphtheria, 49.6% for pertussis, 82.2% for tetanus, 76.7% for HepB and 97.7% for Hib (anti-PRP ≥ 0.15 µg/ml). The Quinvaxem® vaccine was well tolerated and has a low rate of adverse events. Quinvaxem® given at 2, 3 and 4 months of age was immunogenic and safe for primary immunization among infants in Vietnam.

  2. Heparin-binding epidermal growth factor-like growth factor/diphtheria toxin receptor expression by acute myeloid leukemia cells.

    PubMed

    Vinante, F; Rigo, A; Papini, E; Cassatella, M A; Pizzolo, G

    1999-03-01

    Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an EGF family member expressed by numerous cell types that binds to EGF receptor 1 (HER-1) or 4 (HER-4) inducing mitogenic and/or chemotactic activities. Membrane-bound HB-EGF retains growth activity and adhesion capabilities and the unique property of being the receptor for diphtheria toxin (DT). The interest in studying HB-EGF in acute leukemia stems from these mitogenic, chemotactic, and receptor functions. We analyzed the expression of HB-EGF in L428, Raji, Jurkat, Karpas 299, L540, 2C8, HL-60, U937, THP-1, ML-3, and K562 cell lines and in primary blasts from 12 acute myeloid leukemia (AML) cases, by reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blot and by the evaluation of sensitivity to DT. The release of functional HB-EGF was assessed by evaluation of its proliferative effects on the HB-EGF-sensitive Balb/c 3T3 cell line. HB-EGF was expressed by all myeloid and T, but not B (L428, Raji), lymphoid cell lines tested, as well as by the majority (8 of 12) of ex vivo AML blasts. Cell lines (except for the K562 cell line) and AML blasts expressing HB-EGF mRNA underwent apoptotic death following exposure to DT, thus demonstrating the presence of the HB-EGF molecule on their membrane. Leukemic cells also released a fully functional HB-EGF molecule that was mitogenic for the Balb/c 3T3 cell line. Factors relevant to the biology of leukemic growth, such as tumor necrosis factor-alpha (TNF-alpha), 1alpha,25-(OH)2D3, and especially all-trans retinoic acid (ATRA), upregulated HB-EGF mRNA in HL-60 or ML-3 cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced HB-EGF mRNA and acquisition of sensitivity to DT in one previously HB-EGF-negative leukemia case. Moreover, the U937 and Karpas 299 cell lines expressed HER-4 mRNA. This work shows that HB-EGF is a growth factor produced by primary leukemic cells and regulated by ATRA, 1alpha, 25-(OH)2D3, and GM-CSF.

  3. The protective effect of immunisation against diphtheria, pertussis and tetanus (DPT) in relation to sudden infant death syndrome.

    PubMed

    Essery, S D; Raza, M W; Zorgani, A; MacKenzie, D A; James, V S; Weir, D M; Busuttil, A; Hallam, N; Blackwell, C

    1999-08-01

    Epidemiological evidence indicates infants immunised against diphtheria, pertussis and tetanus (DPT) are at decreased risk of sudden infant death syndrome (SIDS). Asymptomatic whooping cough and pyrogenic toxins of Staphylococcus aureus have been implicated in the aetiology of SIDS. The objectives of the present study were: (1) to determine if the DPT vaccine induced antibodies cross-reactive with the staphylococcal toxins; (2) to determine if antibodies to the pertussis toxin (PT) and the staphylococcal toxins were present in the sera of women during late pregnancy; (3) to examine the effects of infant immunisation on levels of antibodies to PT and the staphylococcal toxins; (4) to assess the effects of changes in immunisation schedules in the UK on the incidence and age distribution of SIDS. Enzyme-linked immunosorbent assays (ELISA) were used to measure binding of rabbit or human IgG to the DPT vaccine, PT, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins A (SEA), B (SEB) and C (SEC). Neutralisation activity of anti-DPT serum was assessed by a bioassay for induction of nitric oxide from human monocytes by the staphylococcal toxins. Anti-DPT serum bound to the DPT vaccine, PT and each of the staphylococcal toxins. It also reduced the ability of the four toxins to induce nitric oxide from monocytes. In pregnant women, levels of IgG to PT, SEC and TSST-1 decreased significantly in relation to increasing weeks of gestation while antibodies to SEA and SEB increased. In infants' sera there were significant correlations between levels of IgG bound to DPT and IgG bound to PT, TSST-1 and SEC but not SEA or SEB. Antibody levels to the toxins in infants declined with age; sera from infants < or = 2 months of age had higher levels of IgG bound to the toxins than those older than 2 months. This pattern was observed for infants whose immunisation schedules began at 2 months of age or 3 months of age. The decrease in IgG bound to the toxins was, however

  4. A clinical trial examining the effect of increased total CRM(197) carrier protein dose on the antibody response to Haemophilus influenzae type b CRM(197) conjugate vaccine.

    PubMed

    Usonis, Vytautas; Bakasenas, Vytautas; Lockhart, Stephen; Baker, Sherryl; Gruber, William; Laudat, France

    2008-08-18

    CRM(197) is a carrier protein in certain conjugate vaccines. When multiple conjugate vaccines with the same carrier protein are administered simultaneously, reduced response to vaccines and/or antigens related to the carrier protein may occur. This study examined responses of infants who, in addition to diphtheria toxoid/tetanus toxoid/acellular pertussis vaccine (DTaP) received either diphtheria CRM(197)-based Haemophilus influenzae type b conjugate vaccine (HbOC) or HbOC and a diphtheria CRM(197)-based combination 9-valent pneumococcal conjugate vaccine/meningococcal group C conjugate vaccine. Administration of conjugate vaccines with CRM(197) carrier protein load >50 microg did not reduce response to CRM(197) conjugate vaccines or immunogenicity to immunologically cross-reactive diphtheria toxoid.

  5. Novel hemin binding domains in the Corynebacterium diphtheriae HtaA protein interact with hemoglobin and are critical for heme iron utilization by HtaA.

    PubMed

    Allen, Courtni E; Schmitt, Michael P

    2011-10-01

    The human pathogen Corynebacterium diphtheriae utilizes hemin and hemoglobin as iron sources for growth in iron-depleted environments. The use of hemin iron in C. diphtheriae involves the dtxR- and iron-regulated hmu hemin uptake locus, which encodes an ABC hemin transporter, and the surface-anchored hemin binding proteins HtaA and HtaB. Sequence analysis of HtaA and HtaB identified a conserved region (CR) of approximately 150 amino acids that is duplicated in HtaA and present in a single copy in HtaB. The two conserved regions in HtaA, designated CR1 and CR2, were used to construct glutathione S-transferase (GST) fusion proteins (GST-CR1 and GST-CR2) to assess hemin binding by UV-visual spectroscopy. These studies showed that both domains were able to bind hemin, suggesting that the conserved sequences are responsible for the hemin binding property previously ascribed to HtaA. HtaA and the CR2 domain were also shown to be able to bind hemoglobin (Hb) by the use of an enzyme-linked immunosorbent assay (ELISA) method in which Hb was immobilized on a microtiter plate. The CR1 domain exhibited a weak interaction with Hb in the ELISA system, while HtaB showed no significant binding to Hb. Competitive binding studies demonstrated that soluble hemin and Hb were able to inhibit the binding of HtaA and the CR domains to immobilized Hb. Moreover, HtaA was unable to bind to Hb from which the hemin had been chemically removed. Alignment of the amino acid sequences of CR domains from various Corynebacterium species revealed several conserved residues, including two highly conserved tyrosine (Y) residues and one histidine (H) residue. Site-directed mutagenesis studies showed that Y361 and H412 were critical for the binding to hemin and Hb by the CR2 domain. Biological assays showed that Y361 was essential for the hemin iron utilization function of HtaA. Hemin transfer experiments demonstrated that HtaA was able to acquire hemin from Hb and that hemin bound to HtaA could be

  6. The ChrA response regulator in Corynebacterium diphtheriae controls hemin-regulated gene expression through binding to the hmuO and hrtAB promoter regions.

    PubMed

    Burgos, Jonathan M; Schmitt, Michael P

    2012-04-01

    Corynebacterium diphtheriae, the etiologic agent of diphtheria, utilizes heme and hemoglobin (Hb) as iron sources for growth. Heme-iron utilization involves HmuO, a heme oxygenase that degrades cytosolic heme, resulting in the release of heme-associated iron. Expression of the hmuO promoter is under dual regulation, in which transcription is repressed by DtxR and iron and activated by a heme source, such as hemin or Hb. Hemin-dependent activation is mediated primarily by the ChrAS two-component system, in which ChrS is a putative heme-responsive sensor kinase while ChrA is proposed to serve as a response regulator that activates transcription. It was recently shown that the ChrAS system similarly regulates the hrtAB genes, which encode an ABC transporter involved in the protection of C. diphtheriae from hemin toxicity. In this study, we characterized the phosphorelay mechanism in the ChrAS system and provide evidence for the direct regulation of the hmuO and hrtAB promoters by ChrA. A fluorescence staining method was used to show that ChrS undergoes autophosphorylation and that the phosphate moiety is subsequently transferred to ChrA. Promoter fusion studies identified regions upstream of the hmuO and hrtAB promoters that are critical for the heme-dependent regulation by ChrA. Electrophoretic mobility shift assays revealed that ChrA specifically binds at the hmuO and hrtAB promoter regions and that binding is phosphorylation dependent. A phosphorylation-defective mutant of ChrA [ChrA(D50A)] exhibited significantly diminished binding to the hmuO promoter region relative to that of wild-type ChrA. DNase I footprint analysis further defined the sequences in the hmuO and hrtAB promoters that are involved in ChrA binding, and this analysis revealed that the DtxR binding site at the hmuO promoter partially overlaps the binding site for ChrA. DNase I protection studies as well as promoter fusion analysis suggest that ChrA and DtxR compete for binding at the hmuO promoter

  7. Fast, antigen-saving multiplex immunoassay to determine levels and avidity of mouse serum antibodies to pertussis, diphtheria, and tetanus antigens.

    PubMed

    Stenger, Rachel M; Smits, Mieke; Kuipers, Betsy; Kessen, Sabine F M; Boog, Claire J P; van Els, Cécile A C M

    2011-04-01

    To enhance preclinical evaluation of serological immune responses to the individual diphtheria, tetanus, and pertussis (DTP) components of DTP combination vaccines, a fast hexavalent bead-based method was developed. This multiplex immunoassay (MIA) can simultaneously determine levels of specific mouse serum IgG antibodies to P antigens P.69 pertactin (P.69 Prn), filamentous hemagglutinin (FHA), pertussis toxin (Ptx), and combined fimbria type 2 and 3 antigens (Fim2/3) and to diphtheria toxin (Dtx) and tetanus toxin (TT) in a single well. The mouse DTP MIA was shown to be specific and sensitive and to correlate with the six single in-house enzyme-linked immunosorbent assays (ELISAs) for all antigens. Moreover, the MIA was expanded to include avidity measurements of DTP antigens in a multivalent manner. The sensitivities of the mouse DTP avidity MIA per antigen were comparable to those of the six individual in-house avidity ELISAs, and good correlations between IgG concentrations obtained by both methods for all antigens tested were shown. The regular and avidity mouse DTP MIAs were reproducible, with good intra- and interassay coefficients of variability (CV) for all antigens. Finally, the usefulness of the assay was demonstrated in a longitudinal study of the development and avidity maturation of specific IgG antibodies in mice having received different DTP vaccines. We conclude that the hexaplex mouse DTP MIA is a specific, sensitive, and high-throughput alternative for ELISA to investigate the quantity and quality of serological responses to DTP antigens in preclinical vaccine studies.

  8. Pulmonary immunization of guinea pigs with diphtheria CRM-197 antigen as nanoparticle aggregate dry powders enhance local and systemic immune responses.

    PubMed

    Muttil, Pavan; Pulliam, Brian; Garcia-Contreras, Lucila; Fallon, John Kevin; Wang, Chenchen; Hickey, Anthony James; Edwards, David A

    2010-12-01

    This study establishes the immune response elicited in guinea pigs after pulmonary and parenteral immunizations with diphtheria CRM-197 antigen (CrmAg). Several spray-dried powders of formalin-treated/untreated CrmAg nanoaggregates with L-leucine were delivered to the lungs of guinea pigs. A control group consisting of alum with adsorbed CrmAg in saline was administered by intramuscular injection. Animals received three doses of powder vaccines containing 20 or 40 μg of CrmAg. The serum IgG titers were measured for 16 weeks after the initial immunization; IgA titers were measured at the time of sacrifice in the broncho-alveolar lavage fluid. Further, toxin neutralization tests in naïve guinea pigs were performed for a few select serum samples. Histopathology of the lung tissues was conducted to evaluate inflammation or injury to the lung tissues. While the highest titer of serum IgG antibody was observed in guinea pigs immunized by the intramuscular route, those animals immunized with dry powder formulation by the pulmonary route, and without the adjuvant alum, exhibited high IgA titers. A pulmonary administered dry powder, compared to parenteral immunization, conferred complete protection in the toxin neutralization test. Mild inflammation was observed in lung tissues of animals receiving dry powder vaccines by the pulmonary route. Thus, administering novel CrmAg as dry powders to the lungs may be able to overcome some of the disadvantages observed with the existing diphtheria vaccine which is administered by the parenteral route. In addition, these powders will have the advantage of eliciting a high mucosal immune response in the lungs without using traditional adjuvants.

  9. A Belgian Serosurveillance/Seroprevalence Study of Diphtheria, Tetanus and Pertussis Using a Luminex xMAP Technology-Based Pentaplex

    PubMed Central

    Caboré, Raissa Nadège; Piérard, Denis; Huygen, Kris

    2016-01-01

    Serosurveillance and seroprevalence studies are an essential tool to monitor vaccine-preventable diseases. We have developed a magnetic bead-based pentaplex immunoassay (MIA) for the simultaneous detection of IgG antibodies against diphtheria toxin (DT), tetanus toxin (TT), pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (Prn). The in-house pentaplex MIA showed a good correlation with commercial ELISAs with correlation coefficients between 0.89 for PT and 0.98 for TT. Intra- and inter-assay variability was <10%. A total of 670 anonymized serum samples collected in 2012 in Belgian adults (ages 20–29.9 years) were analyzed. Geometric mean concentrations (GMC) were 0.2 (0.13–0.29) IU/mL for DT, 0.63 (0.45–0.82) IU/mL for TT, 3.9 (2.6–5.8) IU/mL for PT, 16.3 (11.7–22.7) IU/mL for FHA and 15.4 (10.1–23.6) IU/mL for Prn. Antibody concentrations were below the protective level of 0.1 IU/mL in 26.4% of the sera for DT and in 8.6% of the sera for TT. Anti-PT IgG concentrations indicative of recent pertussis infection (>125 IU/mL) were detected in 1.2% of the subjects. High anti-PT antibodies were not correlated with high antibodies against any of the four other vaccine antigens. This pentaplex MIA will be used for a new large-scale Belgian serosurveillance/seroprevalence study of diphtheria, tetanus and pertussis. PMID:27171114

  10. Travelers' Health: Diphtheria

    MedlinePlus

    ... at 770-488-7100. An antibiotic (erythromycin or penicillin) should be used to eliminate the causative organisms, ... cardiac monitoring) is required. Antimicrobial prophylaxis (erythromycin or penicillin) is recommended for close contacts of patients. PREVENTION ...

  11. Statistics on Diphtheria

    MedlinePlus

    ... Immunization, Vaccines and Biologicals Immunization surveillance, assessment and monitoring Disease surveillance and Burden Quick Links Sitemap Home Health topics Data Media centre Publications Countries Programmes and ...

  12. Safety and reactogenicity of the combined diphtheria-tetanus-acellular pertussis-inactivated poliovirus-Haemophilus influenzae type b (DTPa-IPV/Hib) vaccine in healthy Vietnamese toddlers: An open-label, phase III study

    PubMed Central

    Anh, Dang Duc; Van Der Meeren, Olivier; Karkada, Naveen; Assudani, Deepak; Yu, Ta-Wen; Han, Htay Htay

    2016-01-01

    abstract The introduction of combination vaccines plays a significant role in increasing vaccine acceptance and widening vaccine coverage. Primary vaccination against diphtheria, tetanus, pertussis, poliomyelitis and Haemophilus influenza type b (Hib) diseases has been implemented in Vietnam. In this study we evaluated the safety and reactogenicity of combined diphtheria-tetanus-pertussis-inactivated polio (DTPa-IPV)/Hib vaccine when administered as a booster dose in 300 healthy Vietnamese children <2 years of age (mean age: 15.8 months). During the 4-day follow-up period, pain (31.7%) and redness (27.3%) were the most frequent solicited local symptoms. Pain (2%) was also the most frequent grade 3 local symptom. One subject reported 2 serious adverse events that were not causally related to the study vaccine. DTPa-IPV/Hib conjugate vaccine was well tolerated as a booster dose in healthy Vietnamese children aged <2 years. PMID:26337197

  13. Safety and reactogenicity of the combined diphtheria-tetanus-acellular pertussis-inactivated poliovirus-Haemophilus influenzae type b (DTPa-IPV/Hib) vaccine in healthy Vietnamese toddlers: An open-label, phase III study.

    PubMed

    Anh, Dang Duc; Van Der Meeren, Olivier; Karkada, Naveen; Assudani, Deepak; Yu, Ta-Wen; Han, Htay Htay

    2016-03-03

    The introduction of combination vaccines plays a significant role in increasing vaccine acceptance and widening vaccine coverage. Primary vaccination against diphtheria, tetanus, pertussis, poliomyelitis and Haemophilus influenza type b (Hib) diseases has been implemented in Vietnam. In this study we evaluated the safety and reactogenicity of combined diphtheria-tetanus-pertussis-inactivated polio (DTPa-IPV)/Hib vaccine when administered as a booster dose in 300 healthy Vietnamese children <2 years of age (mean age: 15.8 months). During the 4-day follow-up period, pain (31.7%) and redness (27.3%) were the most frequent solicited local symptoms. Pain (2%) was also the most frequent grade 3 local symptom. One subject reported 2 serious adverse events that were not causally related to the study vaccine. DTPa-IPV/Hib conjugate vaccine was well tolerated as a booster dose in healthy Vietnamese children aged <2 years.

  14. The ABC transporter HrtAB confers resistance to hemin toxicity and is regulated in a hemin-dependent manner by the ChrAS two-component system in Corynebacterium diphtheriae.

    PubMed

    Bibb, Lori A; Schmitt, Michael P

    2010-09-01

    Corynebacterium diphtheriae, the causative agent of the severe respiratory disease diphtheria, utilizes hemin and hemoglobin as iron sources for growth in iron-depleted environments. Because of the toxicity of high levels of hemin and iron, these compounds are often tightly regulated in bacterial systems. In this report, we identify and characterize the C. diphtheriae hrtAB genes, which encode a putative ABC type transporter involved in conferring resistance to the toxic effects of hemin. Deletion of the hrtAB genes in C. diphtheriae produced increased sensitivity to hemin, which was complemented by a plasmid harboring the cloned hrtAB locus. The HrtAB system was not involved in the uptake and use of hemin as an iron source. The hrtAB genes are located on the C. diphtheriae genome upstream from the chrSA operon, which encodes a previously characterized two-component signal transduction system that regulates gene expression in a heme-dependent manner. The hrtB promoter is activated by the ChrAS system in the presence of hemin or hemoglobin, and mutations in the chrSA genes abolish heme-activated expression from the hrtB promoter. It was also observed that transcription from the hrtB promoter is reduced in a dtxR deletion mutant, suggesting that DtxR is required for optimal expression of hrtAB. Previous studies proposed that the ChrS sensor kinase may be responsive to an environmental signal, such as hemin. We show that specific point mutations in the ChrS N-terminal transmembrane domain result in a reduced ability to activate the hrtB promoter in the presence of a heme source, suggesting that this putative sensor region is essential for the detection of a signal produced in response to hemin exposure. This study shows that the HrtAB system is required for protection from hemin toxicity and that expression of the hrtAB genes is regulated by the ChrAS two-component system. This study demonstrates a direct correlation between the detection of heme or a heme

  15. Pattern of functional antibody activity against Haemophilus influenzae type B (Hib) in infants immunized with diphtheria-tetanus-pertussis/Hib Brazilian combination vaccine.

    PubMed

    Matos, D C S; Silva, A M V; Neves, P C C; Martins, R M; Homma, A; Marcovistz, R

    2009-12-01

    We evaluated the functional activity of Haemophilus influenzae B (Hib) antibodies elicited in a group of infants immunized with the diphtheria-tetanus-pertussis vaccine combined with an Hib vaccine produced totally in Brazil after technological transfer of Hib vaccine production from Glaxo SmithKline, Belgium. Blood samples from immunized infants (N = 985) were collected for the determination of Hib antibodies. Total Ig and IgM and IgG subclasses of antibodies against polyribosyl ribitol phosphate (PRP) were analyzed by ELISA. Almost all vaccinees (97.56%, 961/985) developed a strong anti-PRP IgG antibody response (>or=1.0 microg/mL), while an anti-PRP IgM response was observed in 64.24% (634/985) of them (>or=0.15 microg/mL). Only 18.88% (186/985) of the infants in the group with high PRP antibody IgG concentrations (>or=1.0 microg/mL) developed a high IgM antibody response. Anti-PRP IgG antibody levels were significantly higher than anti-PRP IgM. These results demonstrate the predominance of IgG antibodies over IgM antibodies in response to PRP, with a ratio of 17:1. IgG antibodies were predominantly of the IgG1 subclass. An increase in IgG avidity was also observed during the course of immunization.

  16. Guinea pig line 10 hepatocarcinoma model: characterization of monoclonal antibody and in vivo effect of unconjugated antibody and antibody conjugated to diphtheria toxin A chain.

    PubMed

    Bernhard, M I; Foon, K A; Oeltmann, T N; Key, M E; Hwang, K M; Clarke, G C; Christensen, W L; Hoyer, L C; Hanna, M G; Oldham, R K

    1983-09-01

    Monoclonal antibodies were raised against the guinea pig line 10 (L10) hepatocarcinoma, and an IgG1-producing hybridoma (D3) was selected for further study. D3 is a true monoclonal antibody as demonstrated by two-dimensional gel electrophoresis. Radioimmunoassays on live cells revealed no cross-reactivity with normal tissues or with the line 1 hepatocarcinoma which was used as a control. Membrane immunofluorescence assays demonstrated similar specificity. Immunoperoxidase staining of cryostat sections of tumor and normal tissues of both adult animals and fetuses showed that the D3 monoclonal antibody reacted primarily with the L10 tumor, but some cross-reactivity with smooth muscle, placenta, fetal skeletal muscle, and fetal liver was also demonstrated. Radioimmunoprecipitation of detergent extracts of iodinated L10 cells showed that the antigen is present on the cell surface as a dimer of Mr 290,000 (unit size, Mr 148,000). Therapy studies with unconjugated D3 antibody demonstrated a minor dose-dependent effect on tumor growth. D3 antibody conjugated to the A chain of diphtheria toxin (10(-7) M) was cytotoxic to 100% of L10 cells in vitro. Animals treated with a single 1-mg i.v. injection of this immunoconjugate on Day 7 following the intradermal injection of 10(5) tumor cells demonstrated a highly significant inhibition of tumor growth compared to control animals and those treated with unconjugated antibody.

  17. Evaluation of a new syringe presentation of reduced-antigen content diphtheria, tetanus, and acellular pertussis vaccine in healthy adolescents - A single blind randomized trial

    PubMed Central

    Pavia-Ruz, Noris; Abarca, Katia; Lepetic, Alejandro; Cervantes-Apolinar, Maria Yolanda; Hardt, Karin; Jayadeva, Girish; Kuriyakose, Sherine; Han, Htay Htay; de la O, Manuel

    2015-01-01

    Reduced-antigen-content diphtheria-tetanus-acellular pertussis (dTpa) vaccine, Boostrix™, is indicated for booster vaccination of children, adolescents and adults. The original prefilled disposable dTpa syringe presentation was recently replaced by another prefilled-syringe presentation with latex-free tip-caps and plunger-stoppers. 671 healthy adolescents aged 10–15 years who had previously received 5 or 6 previous DT(P)/dT(pa) vaccine doses, were randomized (1:1) to receive dTpa booster, injected using the new (dTpa-new) or previous syringe (dTpa-previous) presentations. Immunogenicity was assessed before and 1-month post-booster vaccination; safety/reactogenicity were assessed during 31-days post-vaccination. Non-inferiority of dTpa-new versus dTpa-previous was demonstrated for all antigens (ULs 95% CIs for GMC ratios ranged between 1.03-1.13). 1-month post-booster, immune responses were in similar ranges for all antigens with both syringe presentations. dTpa delivered using either syringe presentation was well-tolerated. These clinical results complement the technical data and support the use of the new syringe presentation to deliver the dTpa vaccine. PMID:26075317

  18. Role of Acidic Residues in Helices TH8–TH9 in Membrane Interactions of the Diphtheria Toxin T Domain

    PubMed Central

    Ghatak, Chiranjib; Rodnin, Mykola V.; Vargas-Uribe, Mauricio; McCluskey, Andrew J.; Flores-Canales, Jose C.; Kurnikova, Maria; Ladokhin, Alexey S.

    2015-01-01

    The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8–TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8–TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions. PMID:25875295

  19. Heat-induced transcription of diphtheria toxin A or its variants, CRM176 and CRM197: implications for pancreatic cancer gene therapy.

    PubMed

    Fogar, P; Navaglia, F; Basso, D; Zambon, C-F; Moserle, L; Indraccolo, S; Stranges, A; Greco, E; Fadi, E; Padoan, A; Pantano, G; Sanzari, M C; Pedrazzoli, S; Montecucco, C; Plebani, M

    2010-01-01

    Vectors combining the heat shock proteins (HSPs) promoter with the catalytic subunit A of the diphtheria toxin (DTA) or its variants, cross-reacting material (CRM) 176 and 197, were engineered to investigate the effect of bacterial toxins on pancreatic cancer (PC) cells. Three heat-inducible enhanced green fluorescent protein (eGFP)-expression vectors were obtained: V1 (91% homology to HSPA6), V2 (five heat shock elements upstream the minimal HSPA6 promoter) and V3 (V1 and V2 combined). The highest eGFP transcription and translation levels were found in V3 transfected PC cells. The V3 promoter was used to control DTA, CRM176 and CRM197 expression, treatment response being investigated in four PC cell lines. DTAwt or CRM176 transfected cell growth was completely arrested after heat shock. CRM197 toxin presumed to be inactive, caused mild distress at 37 degrees C and induced a 25-50% reduction in cell growth after heat shock. Preliminary in vivo findings showed that heat treatment arrests tumor growth in DTA197 stably transfected PSN1 cells. In conclusion, the efficient HSP promoter identified in this study may be extremely useful in controlling the transcription of toxins such as CRM197, which have lethal dose-related effects, and may thus be a promising tool in PC gene therapy in vivo.

  20. Targeted therapy to the IL-2R using diphtheria toxin and caspase-3 fusion proteins modulates Treg and ameliorates inflammatory colitis.

    PubMed

    Yarkoni, Shai; Sagiv, Yuval; Kaminitz, Ayelet; Farkas, Daniel L; Askenasy, Nadir

    2009-10-01

    Pathogenic lymphocytes in the enteric wall of inflammatory bowel disease patients display various abnormalities, including reduced sensitivity to apoptosis. We evaluated a therapeutic approach to elimination of cytotoxic cells, using two IL-2 fusion proteins, a diphtheria toxin (IL2-DT) and a caspase-3 (IL2-cas) conjugate. In models of acute (dextran sodium sulfate and trinitrobenzene sulfonic acid) and chronic (dextran sodium sulfate) toxic colitis, therapeutic doses of the fusion proteins improved survival and prevented colon shortening. While both chimeric proteins eradicated CD4(+)CD25(+)Foxp3(+) T cells in mesenteric LN, IL2-DT caused severe lymphopenia. In contrast, IL2-cas was equally protective and increased fractional expression of Foxp3. Similar effects of the fusion proteins were observed in healthy mice: IL2-DT caused lymphopenia and IL2-cas increased fractional expression of FoxP3. The fusion proteins induced apoptosis in CD25(+) T cells in vitro, with lower toxicity of IL2-cas to Foxp3(+) T cells. These data infer that targeted depletion of cells expressing the IL-2 receptor has therapeutic potential in models of inflammatory colitis, despite depletion of CD25(+) Treg. The IL2-cas fusion protein is particularly relevant to inflammatory bowel disease, as direct internalization of toxic moieties overcomes multiple pathways of resistance to apoptosis of colitogenic T cells.

  1. Both Corynebacterium diphtheriae DtxR(E175K) and Mycobacterium tuberculosis IdeR(D177K) are dominant positive repressors of IdeR-regulated genes in M. tuberculosis.

    PubMed

    Manabe, Yukari C; Hatem, Christine L; Kesavan, Anup K; Durack, Justin; Murphy, John R

    2005-09-01

    The diphtheria toxin repressor (DtxR) is an important iron-dependent transcriptional regulator of known virulence genes in Corynebacterium diphtheriae. The mycobacterial iron-dependent repressor (IdeR) is phylogenetically closely related to DtxR, with high amino acid similarity in the DNA binding and metal ion binding site domains. We have previously shown that an iron-insensitive, dominant-positive dtxR(E175K) mutant allele from Corynebacterium diphtheriae can be expressed in Mycobacterium tuberculosis and results in an attenuated phenotype in mice. In this paper, we report the M. tuberculosis IdeR(D177K) strain that has the cognate point mutation. We tested four known and predicted IdeR-regulated gene promoters (mbtI, Rv2123, Rv3402c, and Rv1519) using a promoterless green fluorescent protein (GFP) construct. GFP expression from these promoters was abrogated under low-iron conditions in the presence of both IdeR(D177K) and DtxR(E175K), a result confirmed by reverse transcription-PCR. The IdeR regulon can be constitutively repressed in the presence of an integrated copy of ideR containing this point mutation. These data also suggest that mutant IdeR(D177K) has a mechanism similar to that of DtxR(E175K); iron insensitivity occurs as a result of SH3-like domain binding interactions that stabilize the intermediate form of the repressor after ancillary metal ion binding. This construct can be used to elucidate further the IdeR regulon and its virulence genes and to differentiate these from genes regulated by SirR, which does not have this domain.

  2. A CpG-containing oligodeoxynucleotide as an efficient adjuvant counterbalancing the Th1/Th2 immune response in diphtheria-tetanus-pertussis vaccine.

    PubMed

    Sugai, Toshiyuki; Mori, Masaaki; Nakazawa, Masatoshi; Ichino, Motohide; Naruto, Takuya; Kobayashi, Naoki; Kobayashi, Yoshinori; Minami, Mutsuhiko; Yokota, Shumpei

    2005-11-16

    Adjuvants in vaccines are immune stimulants that play an important role in the induction of effective and appropriate immune responses to vaccine component(s). Diphtheria-tetanus-pertussis (DPT) vaccine contains not only aluminum hydrate (alum) to enhance the immune response to the vaccine ingredients, but also, both for that purpose and as a principal ingredient, pertussis toxin (PT). However, both adjuvants strongly promote T helper (Th) 2 type immune responses. Th1 and Th2 type immune responses are counterbalanced in vivo, and a Th2-prone immune response is not effective against intracellular infections but promotes IgE production, which is related to allergic disease. In this study, we used the CpG motif contained in oligodeoxynucleotide (CpG-ODN), which has an adjuvant effect and also induces the Th1 response, as an adjuvant to this vaccine, and we investigated its adjuvanticity and its potential to modulate immune responses to DPT vaccine. Administration of DPT vaccine with CpG-ODN (DPT-alum/ODN) to mice significantly reduced the total IgE levels and increased the anti-PT specific IgG2a titer in serum, in comparison with ordinary DPT vaccine (DPT-alum). Moreover, we investigated the antibody response to orally administrated ovalbumin (OVA) after vaccine administration. In the DPT-alum/ODN-administered group, the OVA specific IgE production in serum greatly decreased in comparison with that in the DPT-alum-administered group. These data indicate that CpG-ODN was not useful only as an efficient vaccine adjuvant but also shifted the immune responses substantially toward Th1 and modulated the Th1/Th2 immune response in DPT vaccine. These data suggested new applications of CpG-ODN as adjuvants in DPT vaccine.

  3. Structures of the Substrate-free and Product-bound Forms of HmuO, a Heme Oxygenase from Corynebacterium diphtheriae

    PubMed Central

    Unno, Masaki; Ardèvol, Albert; Rovira, Carme; Ikeda-Saito, Masao

    2013-01-01

    Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe3+-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe3+-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe3+-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe3+-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit. PMID:24106279

  4. Role of active-site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C-S lyase.

    PubMed

    Astegno, Alessandra; Allegrini, Alessandra; Piccoli, Stefano; Giorgetti, Alejandro; Dominici, Paola

    2015-01-01

    In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C-S lyase from Corynebacterium diphtheriae is a pyridoxal 5'-phosphate-dependent enzyme in the transsulfuration pathway that catalyzes the α,β-elimination of sulfur-containing amino acids, such as L-cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L-methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active-site residues, Y55F and Y114F, were characterized by UV-visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady-state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130-fold decrease in K(d)(PLP) at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP-binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of L-cystathionine, and causes a change in substrate specificity towards L-serine and O-acetyl-L-serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C-S lyase, which seems to be regulated by active-site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors.

  5. Effect of tetanus-diphtheria (Td) vaccine on immune response to hepatitis B vaccine in healthy individuals with insufficient immune response

    PubMed Central

    Salehi, Maryam; Haghighat, Abbas; Salehi, Hassan; Taleban, Roya; Salehi, Marzieh; Kalbasi, Nader; Moafi, Mohammad; Salehi, Mohammad Mahdi

    2015-01-01

    Background: Hepatitis B virus (HBV) fails to produce appropriate immune responses in some healthy individuals; thus, different strategies have been adopted to promote immune responses. The current study aimed at evaluating the efficacy of HBV vaccine coadministered with tetanus-diphtheria (Td) vaccine compared with HBV vaccine in healthy individuals through measuring hepatitis B surface antibody (HBsAb) levels. Materials and Methods: This was a randomized controlled clinical trial, which was implemented in Isfahan, Isfahan Province (Iran) in 2013. One hundred and forty healthy individuals, whose HBsAb titers were less than 10 IU/L were recruited. The subjects were randomly assigned to either in intervention or control trials. The control group received 40 μg of recombinant HBV vaccines intramuscularly injected at 0, 1, and 6 months; however, the intervention group was simultaneously vaccinated by Td with the first dose of HBV vaccine. HBV antibody levels (titer) were measured before the vaccination and 6 months after the last vaccination. Results: Antibody titers of the subjects in the intervention and control groups increased from 5.07 ± 2.9 IU/L to 744.45 ± 353.07 IU/L and from 4.45 ± 3.4 IU/L to 589.94 ± 353 IU/L, respectively (both P < 0.001). Also, the mean difference of antibody titer was significantly different between the two groups (P = 0.011). Conclusion: Td vaccination can be applied as a feasible approach to promote efficient and persistent immunity in healthy individuals with insufficient HBsAb titers. PMID:26929760

  6. Influence of maternal vaccination against diphtheria, tetanus, and pertussis on the avidity of infant antibody responses to a pertussis containing vaccine in Belgium.

    PubMed

    Caboré, Raïssa Nadège; Maertens, Kirsten; Dobly, Alexandre; Leuridan, Elke; Van Damme, Pierre; Huygen, Kris

    2017-02-22

    Maternal antibodies induced by vaccination during pregnancy cross the placental barrier and can close the susceptibility gap to pertussis in young infants up to the start of primary immunization. As not only the quantity but also the quality of circulating antibodies is important for protection, we assessed whether maternal immunization affects the avidity of infant vaccine-induced IgG antibodies, in the frame of a prospective clinical trial on pregnancy vaccination in Belgium. Infants born from Tdap (Boostrix®) vaccinated (N = 55) and unvaccinated (N = 26) mothers were immunized with a hexavalent pertussis containing vaccine (Infanrix Hexa®) at 8, 12 and 16 weeks, followed by a fourth dose at 15 months of age. Right before and one month after this fourth vaccine dose, the avidity of IgG antibodies against diphtheria toxin (DT), tetanus toxin (TT), pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (Prn) was determined using 1.5 M ammonium thiocyanate as dissociating agent. In both groups, antibody avidity was moderate for TT, PT, FHA and Prn and low for DT after priming. After a fourth dose, antibody avidity increased significantly to high avidity for TT and PT, whereas it remained moderate for FHA and Prn and low for DT. The avidity correlated positively with antibody level in both study groups, yet not significantly for PT. When comparing both study groups, only PT-specific antibodies showed significantly lower avidity in infants born from vaccinated than from unvaccinated mothers after the fourth vaccine dose. The clinical significance of lower avidity of vaccine induced infant antibodies after maternal vaccination, if any, needs further investigation.

  7. FOLDING OF DIPHTHERIA TOXIN T-DOMAIN IN THE PRESENCE OF AMPHIPOLS AND FLUORINATED SURFACTANTS: TOWARD THERMODYNAMIC MEASUREMENTS OF MEMBRANE PROTEIN FOLDING

    PubMed Central

    Kyrychenko, Alexander; Rodnin, Mykola V.; Vargas-Uribe, Mauricio; Sharma, Shivaji K.; Durand, Grégory; Pucci, Bernard; Popot, Jean-Luc; Ladokhin, Alexey S.

    2011-01-01

    Solubilizing membrane proteins for functional, structural and thermodynamic studies is usually achieved with the help of detergents, which tend to destabilize them, however. Several classes of non-detergent surfactants have been designed as milder substitutes for detergents, most prominently amphipathic polymers called 'amphipols' and fluorinated surfactants. Here we test the potential usefulness of these compounds for thermodynamic studies by examining their effect on conformational transitions of the diphtheria toxin T-domain. The advantage of the T-domain as a model system is that it exists as a soluble globular protein at neutral pH yet is converted into a membrane-competent form by acidification and inserts into the lipid bilayer as part of its physiological action. We have examined the effects of various surfactants on two conformational transitions of the T-domain, thermal unfolding and pH-induced transition to a membrane-competent form. All tested detergent and non-detergent surfactants lowered the cooperativity of the thermal unfolding of the T-domain. The dependence of enthalpy of unfolding on surfactant concentration was found to be least for fluorinated surfactants, thus making them useful candidates for thermodynamic studies. Circular dichroism measurements demonstrate that non-ionic homo-polymeric amphipols (NAhPols), unlike any other surfactants, can actively cause a conformational change of the T-domain. NAhPol-induced structural rearrangements are different from those observed during thermal denaturation and are suggested to be related to the formation of the membrane-competent form of the T-domain. Measurements of vesicle content leakage indicate that interaction with NAhPols not only does not prevent the T-domain from inserting into the bilayer, but it can make bilayer permeabilization even more efficient, whereas the pH-dependence of membrane permeabilization becomes more cooperative. PMID:21945883

  8. Immunogenicity and safety of measles-mumps-rubella and varicella vaccines coadministered with a fourth dose of Haemophilus influenzae type b and Neisseria meningitidis serogroups C and Y-tetanus toxoid conjugate vaccine in toddlers: a pooled analysis of randomized trials.

    PubMed

    Bryant, Kristina; McVernon, Jodie; Marchant, Colin; Nolan, Terry; Marshall, Gary; Richmond, Peter; Marshall, Helen; Nissen, Michael; Lambert, Stephen; Aris, Emmanuel; Mesaros, Narcisa; Miller, Jacqueline

    2012-08-01

    A pooled analysis was conducted of 1257 toddlers who received a fourth dose of Haemophilus influenzae type b-Neisseria meningitidis serogroups C and Y-tetanus toxoid conjugate vaccine (HibMenCY-TT) or Hib conjugate vaccine (Hib polysaccharide conjugated to N. meningitidis outer membrane protein) coadministered with measles-mumps-rubella (MMR) and varicella (VAR) vaccines (NCT00134719/NCT00289783). Noninferiority of immunological responses to MMR and VAR was demonstrated between groups and incidences of MMR- and VAR-specific solicited symptoms were similar, indicating that HibMenCY-TT can be coadministered with MMR and VAR.

  9. Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity

    PubMed Central

    Ruan, Xiaosai; Sack, David A.; Zhang, Weiping

    2015-01-01

    Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent

  10. Preparation, Immunogenicity, and Protective Efficacy, in a Murine Model, of a Conjugate Vaccine Composed of the Polysaccharide Moiety of the Lipopolysaccharide of Vibrio cholerae O139 Bound to Tetanus Toxoid

    PubMed Central

    Boutonnier, Alain; Villeneuve, Sylvain; Nato, Farida; Dassy, Bruno; Fournier, Jean-Michel

    2001-01-01

    The epidemic and pandemic potential of Vibrio cholerae O139 is such that a vaccine against this newly emerged serogroup of V. cholerae is required. A conjugate made of the polysaccharide moiety (O-specific polysaccharide plus core) of the lipopolysaccharide (LPS) of V. cholerae O139 (pmLPS) was prepared by derivatization of the pmLPS with adipic acid dihydrazide and coupling to tetanus toxoid (TT) by carbodiimide-mediated condensation. The immunologic properties of the conjugate were tested using BALB/c mice injected subcutaneously three times at 2 weeks interval and then a fourth time 4 weeks later. Mice were bled 7 days after each injection and then once each month for the following 6 months. LPS and TT antibody levels were determined by enzyme-linked immunosorbent assay using immunoplates coated with either O139 LPS or TT. Both pmLPS and pmLPS-TT conjugate elicited low levels of immunoglobulin M (IgM), peaking 5 weeks after the first immunization. The conjugate elicited high levels of IgG antibodies, peaking 3 months after the first immunization and declining slowly during the following 5 months. TT alone, or as a component of conjugate, induced mostly IgG antibodies. Antibodies elicited by the conjugate recognized both capsular polysaccharide and LPS from V. cholerae O139 and were vibriocidal. They were also protective in the neonatal mouse model of cholera infection. The conjugation of the O139 pmLPS, therefore, enhanced its immunogenicity and conferred T-dependent properties to this polysaccharide. PMID:11292781

  11. Safety and immunogenocity of a novel combined Haemophilus influenzae type b–Neisseria meningitidis serogroups A and C-tetanus-toxoid conjugate vaccine in healthy Chinese children aged 6 months to 5 years old

    PubMed Central

    Hu, Jian-li; Tao, Hong; Li, Jing-xin; Dai, Wei-ming; Song, Bin; Sun, Jin-fang; Liu, Pei; Tang, Jie; Liu, Wen-yu; Wang, Shi-yuan; Zhu, Feng-cai

    2015-01-01

    A novel combined Haemophilus influenzae type b-Neisseria meningitidis serogroups A and C-tetanus-toxoid conjugate vaccine (Hib-MenAC vaccine) has been developed to protect children against diseases caused by Hib, MenA, and MenC. This study investigated the safety and immunogenicity of the Hib-MenAC vaccine administered in 2-dose series to children aged 6–23 months and in a single dose to children aged 2–5 y. A randomized, positive-controlled, non-inferiority clinical trial was conducted for 1200 healthy participants in each age group. Within each age group, participants were randomly allocated to the Hib-MenAC group or the control group at a ratio of 1:1. Adverse reactions were recorded within 28 d after each dose. Blood samples were obtained to assess immunogenicity on day 0 and at 28 d after a complete vaccination course. For the investigational vaccine, the incidence of total adverse reactions in vaccinees aged 6–23 months was 46.8% and that in vaccinees aged 2–5 y was 29.8%. Most adverse reactions were mild or moderate. One non-fatal serious adverse event occurred in the Hib-MenAC group, but was unrelated to vaccination. The seroconversion rate to the 3 components reached 94.0%, and the proportion of vaccinees with rSBA titers ≥ 1:8 and PRP ≥ 0.15 g/mL reached 97.0% in both age groups. The safety and immunogenicity of the Hib-MenAC vaccine were non-inferior when compared to the licensed vaccines. It was concluded that the novel vaccine would be expected to protect children against all of the targeted diseases. PMID:25833163

  12. Safety and immunogenocity of a novel combined Haemophilus influenzae type b-Neisseria meningitidis serogroups A and C-tetanus-toxoid conjugate vaccine in healthy Chinese children aged 6 months to 5 years old.

    PubMed

    Hu, Jian-li; Tao, Hong; Li, Jing-xin; Dai, Wei-ming; Song, Bin; Sun, Jin-fang; Liu, Pei; Tang, Jie; Liu, Wen-yu; Wang, Shi-yuan; Zhu, Feng-cai

    2015-01-01

    A novel combined Haemophilus influenzae type b-Neisseria meningitidis serogroups A and C-tetanus-toxoid conjugate vaccine (Hib-MenAC vaccine) has been developed to protect children against diseases caused by Hib, MenA, and MenC. This study investigated the safety and immunogenicity of the Hib-MenAC vaccine administered in 2-dose series to children aged 6-23 months and in a single dose to children aged 2-5 y. A randomized, positive-controlled, non-inferiority clinical trial was conducted for 1200 healthy participants in each age group. Within each age group, participants were randomly allocated to the Hib-MenAC group or the control group at a ratio of 1:1. Adverse reactions were recorded within 28 d after each dose. Blood samples were obtained to assess immunogenicity on day 0 and at 28 d after a complete vaccination course. For the investigational vaccine, the incidence of total adverse reactions in vaccinees aged 6-23 months was 46.8% and that in vaccinees aged 2-5 y was 29.8%. Most adverse reactions were mild or moderate. One non-fatal serious adverse event occurred in the Hib-MenAC group, but was unrelated to vaccination. The seroconversion rate to the 3 components reached 94.0%, and the proportion of vaccinees with rSBA titers ≥ 1:8 and PRP ≥ 0.15 g/mL reached 97.0% in both age groups. The safety and immunogenicity of the Hib-MenAC vaccine were non-inferior when compared to the licensed vaccines. It was concluded that the novel vaccine would be expected to protect children against all of the targeted diseases.

  13. Mapping the membrane topography of the TH6–TH7 segment of the diphtheria toxin T-domain channel

    PubMed Central

    Wu, Zhengyan; Finkelstein, Alan

    2015-01-01

    Low pH triggers the translocation domain of diphtheria toxin (T-domain), which contains 10 α helices, to insert into a planar lipid bilayer membrane, form a transmembrane channel, and translocate the attached catalytic domain across the membrane. Three T-domain helices, corresponding to TH5, TH8, and TH9 in the aqueous crystal structure, form transmembrane segments in the open-channel state; the amino-terminal region, TH1–TH4, translocates across the membrane to the trans side. Residues near either end of the TH6–TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, it was concluded that the TH6–TH7 segment resides at the cis interface. Now we have examined this segment further, using the substituted-cysteine accessibility method. We constructed a series of 18 mutant T-domains with single cysteine residues at positions in TH6–TH7, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents on the channel conductance. For 10 of the mutants, the reagent caused a change in the single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. For several of these mutants, we verified that the reactions occurred primarily in the open state, rather than in the flicker-closed state. We also established that blocking of the channel by an amino-terminal hexahistidine tag could protect mutants from reaction. Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue’s accessibility from either side. This analysis revealed abrupt changes in cis- versus trans-side accessibility, suggesting that the TH6–TH7 segment forms a constriction that occupies a small portion of the total channel length. We also determined that this constriction is located near the middle of the TH8 helix. PMID:25582482

  14. Stress-Induced Anxiety- and Depressive-Like Phenotype Associated with Transient Reduction in Neurogenesis in Adult Nestin-CreERT2/Diphtheria Toxin Fragment A Transgenic Mice.

    PubMed

    Yun, Sanghee; Donovan, Michael H; Ross, Michele N; Richardson, Devon R; Reister, Robin; Farnbauch, Laure A; Fischer, Stephanie J; Riethmacher, Dieter; Gershenfeld, Howard K; Lagace, Diane C; Eisch, Amelia J

    2016-01-01

    Depression and anxiety involve hippocampal dysfunction, but the specific relationship between these mood disorders and adult hippocampal dentate gyrus neurogenesis remains unclear. In both humans with MDD and rodent models of depression, administration of antidepressants increases DG progenitor and granule cell number, yet rodents with induced ablation of DG neurogenesis typically do not demonstrate depressive- or anxiety-like behaviors. The conflicting data may be explained by the varied duration and degree to which adult neurogenesis is reduced in different rodent neurogenesis ablation models. In order to test this hypothesis we examined how a transient-rather than permanent-inducible reduction in neurogenesis would alter depressive- and anxiety-like behaviors. Transgenic Nestin-CreERT2/floxed diphtheria toxin fragment A (DTA) mice (Cre+DTA+) and littermates (Cre+DTA-; control) were given tamoxifen (TAM) to induce recombination and decrease nestin-expressing stem cells and their progeny. The decreased neurogenesis was transient: 12 days post-TAM Cre+DTA+ mice had fewer DG proliferating Ki67+ cells and fewer DCX+ neuroblasts/immature neurons relative to control, but 30 days post-TAM Cre+DTA+ mice had the same DCX+ cell number as control. This ability of DG neurogenesis to recover after partial ablation also correlated with changes in behavior. Relative to control, Cre+DTA+ mice tested between 12-30 days post-TAM displayed indices of a stress-induced anxiety phenotype-longer latency to consume highly palatable food in the unfamiliar cage in the novelty-induced hypophagia test, and a depression phenotype-longer time of immobility in the tail suspension test, but Cre+DTA+ mice tested after 30 days post-TAM did not. These findings suggest a functional association between adult neurogenesis and stress induced anxiety- and depressive-like behaviors, where induced reduction in DCX+ cells at the time of behavioral testing is coupled with stress-induced anxiety and a

  15. Stress-Induced Anxiety- and Depressive-Like Phenotype Associated with Transient Reduction in Neurogenesis in Adult Nestin-CreERT2/Diphtheria Toxin Fragment A Transgenic Mice

    PubMed Central

    Yun, Sanghee; Donovan, Michael H.; Ross, Michele N.; Richardson, Devon R.; Reister, Robin; Farnbauch, Laure A.; Fischer, Stephanie J.; Riethmacher, Dieter; Gershenfeld, Howard K.; Lagace, Diane C.; Eisch, Amelia J.

    2016-01-01

    Depression and anxiety involve hippocampal dysfunction, but the specific relationship between these mood disorders and adult hippocampal dentate gyrus neurogenesis remains unclear. In both humans with MDD and rodent models of depression, administration of antidepressants increases DG progenitor and granule cell number, yet rodents with induced ablation of DG neurogenesis typically do not demonstrate depressive- or anxiety-like behaviors. The conflicting data may be explained by the varied duration and degree to which adult neurogenesis is reduced in different rodent neurogenesis ablation models. In order to test this hypothesis we examined how a transient–rather than permanent–inducible reduction in neurogenesis would alter depressive- and anxiety-like behaviors. Transgenic Nestin-CreERT2/floxed diphtheria toxin fragment A (DTA) mice (Cre+DTA+) and littermates (Cre+DTA-; control) were given tamoxifen (TAM) to induce recombination and decrease nestin-expressing stem cells and their progeny. The decreased neurogenesis was transient: 12 days post-TAM Cre+DTA+ mice had fewer DG proliferating Ki67+ cells and fewer DCX+ neuroblasts/immature neurons relative to control, but 30 days post-TAM Cre+DTA+ mice had the same DCX+ cell number as control. This ability of DG neurogenesis to recover after partial ablation also correlated with changes in behavior. Relative to control, Cre+DTA+ mice tested between 12–30 days post-TAM displayed indices of a stress-induced anxiety phenotype–longer latency to consume highly palatable food in the unfamiliar cage in the novelty-induced hypophagia test, and a depression phenotype–longer time of immobility in the tail suspension test, but Cre+DTA+ mice tested after 30 days post-TAM did not. These findings suggest a functional association between adult neurogenesis and stress induced anxiety- and depressive-like behaviors, where induced reduction in DCX+ cells at the time of behavioral testing is coupled with stress-induced anxiety

  16. Antibody persistence up to 5 years after vaccination of toddlers and children between 12 months and 10 years of age with a quadrivalent meningococcal ACWY-tetanus toxoid conjugate vaccine.

    PubMed

    Vesikari, Timo; Forsten, Aino; Bianco, Veronique; Van der Wielen, Marie; Miller, Jacqueline M

    2016-01-01

    We studied the persistence of serum bactericidal antibody using rabbit and human complement (rSBA/hSBA, cut-offs 1:8) 5 y after a single dose of meningococcal serogroups A, C, W, Y tetanus toxoid conjugate vaccine (MenACWY-TT) compared with age-appropriate control vaccines in toddlers and children (NCT00427908). Children were previously randomized (3:1) to receive either MenACWY-TT or control vaccine (MenC-CRM197 in 1-<2 y olds; MenACWY-polysaccharide vaccine [Men-PS] in 2-<11 y olds). Subjects with rSBA-MenC titers <1:8 at any time point were revaccinated with MenC conjugate vaccine and discontinued from the study. A repeated measurement statistical model assessed potential selection effects due to drop-outs. At year 5 in MenACWY-TT-vaccinated-toddlers for serogroups A, C, W, and Y respectively, percentages with rSBA titers ≥1:8 were 73.5%, 77.6%, 34.7%, and 42.9%, hSBA ≥1:8 were 35.6%, 91.7%, 82.6% and 80.0%. For MenC-CRM197 recipients, 63.6% had persisting rSBA-MenC titers ≥1:8 and 90.9% had hSBA-MenC ≥1:8 (not significantly different versus MenACWY-TT for either assay: exploratory analyses). In 2-<11 y olds rSBA titers ≥1:8 in MenACWY-TT-vaccinees were 90.8%, 90.8%, 78.6%, and 78.6% and 15.4%, 100%, 0.0%, 7.7% in Men-PS-vaccinees (significantly different for serogroups A, W and Y, exploratory analyses). Serogroups A, W and Y rSBA GMTs were ≥ 26-fold higher in MenACWY-TT-vaccinees. As expected, GMTs modeled at year 5 to assess the impact of subject drop out (mainly for revaccination), appeared lower for serogroup C. No vaccine-related SAEs were reported. Antibody persistence was observed for all serogroups up to 5 y after MenACWY-TT vaccination.

  17. Altered Memory T-Cell Responses to Bacillus Calmette-Guerin and Tetanus Toxoid Vaccination and Altered Cytokine Responses to Polyclonal Stimulation in HIV-Exposed Uninfected Kenyan Infants.

    PubMed

    Garcia-Knight, Miguel A; Nduati, Eunice; Hassan, Amin S; Gambo, Faith; Odera, Dennis; Etyang, Timothy J; Hajj, Nassim J; Berkley, James Alexander; Urban, Britta C; Rowland-Jones, Sarah L

    2015-01-01

    Implementation of successful prevention of mother-to-child transmission of HIV strategies has resulted in an increased population of HIV-exposed uninfected (HEU) infants. HEU infants have higher rates of morbidity and mortality than HIV-unexposed (HU) infants. Numerous factors may contribute to poor health in HEU infants including immunological alterations. The present study assessed T-cell phenotype and function in HEU infants with a focus on memory Th1 responses to vaccination. We compared cross-sectionally selected parameters at 3 and 12 months of age in HIV-exposed (n = 42) and HU (n = 28) Kenyan infants. We measured ex vivo activated and bulk memory CD4 and CD8 T-cells and regulatory T-cells by flow cytometry. In addition, we measured the magnitude, quality and memory phenotype of antigen-specific T-cell responses to Bacillus Calmette-Guerin and Tetanus Toxoid vaccine antigens, and the magnitude and quality of the T cell response following polyclonal stimulation with staphylococcal enterotoxin B. Finally, the influence of maternal disease markers on the immunological parameters measured was assessed in HEU infants. Few perturbations were detected in ex vivo T-cell subsets, though amongst HEU infants maternal HIV viral load positively correlated with CD8 T cell immune activation at 12 months. Conversely, we observed age-dependent differences in the magnitude and polyfunctionality of IL-2 and TNF-α responses to vaccine antigens particularly in Th1 cells. These changes mirrored those seen following polyclonal stimulation, where at 3 months, cytokine responses were higher in HEU infants compared to HU infants, and at 12 months, HEU infant cytokine responses were consistently lower than those seen in HU infants. Finally, reduced effector memory Th1 responses to vaccine antigens were observed in HEU infants at 3 and 12 months and higher central memory Th1 responses to M. tuberculosis antigens were observed at 3 months only. Long-term monitoring of vaccine efficacy

  18. Booster vaccination of pre-school children with reduced-antigen-content diphtheria-tetanus-acellular pertussis-inactivated poliovirus vaccine co-administered with measles-mumps-rubella-varicella vaccine

    PubMed Central

    Ferrera, Giuseppe; Cuccia, Mario; Mereu, Gabriele; Icardi, Giancarlo; Bona, Gianni; Esposito, Susanna; Marchetti, Federico; Messier, Marc; Kuriyakose, Sherine; Hardt, Karin

    2012-01-01

    Background: Pertussis occurs in older children, adolescents and adults due to waning immunity after primary vaccination. Booster vaccination for pre-school children has been recommended in Italy since 1999. In this study (NCT00871000), the immunogenicity, safety and reactogenicity of a booster dose of reduced-antigen content diphtheria-tetanus-acellular pertussis-inactivated poliovirus vaccine (dTpa-IPV; GSK Biologicals Boostrix™-Polio; 3-component pertussis) vs. full-strength DTPa-IPV vaccine (sanofi-pasteur—MSD Tetravac™; 2-component pertussis) was evaluated in pre-school Italian children.   Methods: Healthy children aged 5–6 y primed in a routine vaccination setting with three doses of DTPa-based vaccines were enrolled and randomized (1:1) in this phase IIIb, booster study to receive a single dose of dTpa-IPV or DTPa-IPV; the MMRV vaccine was co-administered. Antibody concentrations/titers against diphtheria, tetanus, pertussis and poliovirus 1–3 were measured before and one month post-booster. Reactogenicity and safety was assessed. Results: 305 subjects were enrolled of whom 303 (dTpa-IPV = 151; DTPa-IPV = 152) received booster vaccination. One month post-booster, all subjects were seroprotected/seropositive for anti-diphtheria, anti-tetanus, anti-PT, anti-FHA and anti-poliovirus 1–3; 99.3% of dTpa-IPV and 60.4% of DTPa-IPV subjects were seropositive for anti-PRN; 98–100% of subjects were seropositive against MMRV antigens post-booster. Pain at the injection site (dTpa-IPV: 63.6%; DTPa-IPV: 63.2%) and fatigue (dTpa-IPV: 26.5%; DTPa-IPV: 23.7%) were the most commonly reported solicited local and general symptoms, during the 4-d follow-up period. No SAEs or fatalities were reported. Conclusions: The reduced-antigen-content dTpa-IPV vaccine was non-inferior to full-strength DTPa-IPV vaccine with respect to immunogenicity. The vaccine was well-tolerated and can be confidently used as a booster dose in pre-school children. PMID:22327497

  19. Antibody persistence after primary and booster doses of a pentavalent vaccine against diphtheria, tetanus, acellular pertussis, inactivated poliovirus, haemophilus influenzae type B vaccine among Thai children at 18-19 months of age.

    PubMed

    Chotpitayasunondh, Tawee; Thisyakorn, Usa; Pancharoen, Chitsanu; Chuenkitmongkol, Sunate; Ortiz, Esteban

    2012-03-01

    The World Health Organization recommends a booster dose of a pertussis-containing vaccine for children aged 1-6 years, preferably during the second year of life. This study assessed the immunogenicity and safety of a pentavalent combination vaccine containing diphtheria, tetanus, acellular pertussis, inactivated poliovirus, and conjugated-Hib polysaccharide antigens, [(DTaP-IPV//PRP-T (Pentaxim)], as a booster at 18-19 months of age. Participants had received primary doses of the same vaccine at 2, 4 and 6 months of age. Antibody concentrations were measured immediately before and one month after the booster dose. Adverse events were evaluated from parental reports. Geometric mean concentrations (GMCs) or titers (GMTs) decreased from post-primary to pre-booster vaccination; however, at least 94.4% of children had protective levels of anti-tetanus (> or = 0.01 IU/ml), antipoliovirus (> or = 81/dil) and anti-PRP (Hib, > or = 0.15 microg/ml) antibodies prior to the booster. Anti-diphtheria antibody titers > or = 0.01 IU/ml were also observed in the majority of children pre-booster. One month after the booster, seroprotection rates were 99.4% for PRP (> or = 1.0 microg/ml), 95.0% for diphtheria (> or = 0.10 IU/ml) and 100% for tetanus (> or = 0.1 IU/ml) and poliovirus types 1, 2, 3 (> or = 81/dil). At least 93.1% of subjects had 4 fold post-booster increases in anti-pertussis antibody titers. GMCs increased from 14.0 to 307.3 EU/ml and from 13.9 to 271.9 EU/ml for anti-PT and anti-FHA, respectively. Anti-PRP GMC increased from 1.2 to 62.2 microg/ml. The booster was well tolerated. A booster dose during the second year of life was safe and induced a strong immune response, indicative of long-term protection.

  20. Immunogenicity and safety of a combined diphtheria, tetanus, 5-component acellular pertussis, inactivated poliomyelitis, Haemophilus type b conjugate vaccine when administered concurrently with a pneumococcal conjugate vaccine: a randomized, open-label, phase 3 study.

    PubMed

    Bernstein, Henry H; Noriega, Fernando

    2011-03-03

    A phase 3 randomized, multicenter study evaluated the safety and immunogenicity of a combined diphtheria, tetanus, 5-component acellular pertussis, inactivated poliomyelitis, Haemophilus influenzae type b conjugate vaccine (DTaP(5)-IPV/Hib) administered at the same visit with 7-valent pneumococcal conjugate vaccine (PCV7, concurrent group) or at separate visits (separated by ≥ 15 days; staggered group). DTaP(5)-IPV/Hib was administered at 2, 4, 6, and 15 months of age, and PCV7 was administered concurrently or at 3, 5, 7, and 16 months of age. The study results found that DTaP(5)-IPV/Hib is safe and immunogenic when given concurrently with 7-valent pneumococcal conjugate vaccine.

  1. Immunogenicity and reactogenicity of two diphtheria-tetanus-acellular pertussis-hepatitis B-inactivated polio virus-Haemophilus influenzae type b vaccines administered at 3, 5 and 11-12 months of age.

    PubMed

    Kilpi, Terhi M; Silfverdal, Sven Arne; Nilsson, Lennart; Syrjänen, Ritva; Belloni, Cesare; Desole, Maria; Triban, Chiara; Storsaeter, Jann; Soila, Maaria; Jacquet, Jeanne-Marie

    2009-01-01

    The use of combination vaccines in the routine childhood program reduces distress to the recipients and is likely to improve uptake rates and timeliness of vaccination but requires careful evaluation and surveillance. The aim of this study was to evaluate the immunogenicity and reactogenicity of two commercial diphtheria-tetanus- acellular pertussis-hepatitis b-inactivated polio virus-Haemophilus influenzae type b (DTaP-HBV-IPV/Hib) combination vaccines when administered to infants at 3, 5 and 11-12 months of age. A total of 494 infants were randomized to receive three doses of either Infanrix hexa (GlaxoSmithKline Biologicals; N = 246) or Hexavac (Sanofi Pasteur MSD; N = 248) in 10 centers in Italy, Finland and Sweden. After the third dose, antibodies to diphtheria, tetanus, polio and Hib were at the protective level in nearly all infants in both groups whereas the proportion of subjects who had achieved the protective concentration of >or=10 mIU/ml to hepatitis B surface antigen was 99.1% (95% CI 96.7-99.9) in the Infanrix hexa group as compared to 94.4% (95% CI 90.4.97.1) in the Hexavac group. Antibody titers to all three polio antigens were highest in Italy and lowest in Finland. Clinically relevant general reactions (such as fever of >39.5 degrees C) were mostly reported in less than 5% of the vaccinees. Three doses of DTaP-HBV-IPV/Hib combination vaccines produced sufficient immune responses in nearly all vaccinees.

  2. Reactivity of murine cytokine fusion toxin, diphtheria toxin390-murine interleukin-3 (DT390-mIL-3), with bone marrow progenitor cells.

    PubMed

    Chan, C H; Blazar, B R; Greenfield, L; Kreitman, R J; Vallera, D A

    1996-08-15

    Myeloid leukemias can express interleukin-3 receptors (IL-3R). Therefore, as an antileukemia drug, a fusion immunotoxin was synthesized consisting of the murine IL-3 (mIL-3) gene spliced to a truncated form of the diphtheria toxin (DT390) gene coding for a molecule that retained full enzymatic activity, but excluded the native binding domain. The DT390-mIL-3 hybrid gene was cloned into a vector under the control of an inducible promote. The fusion protein was expressed in Escherichia coli and then purified from inclusion bodies. The fusion toxin was potent because it inhibited FDC-P1, an IL-3R-expressing murine myelomonocytic tumor line (IC50 = 0.025 nmol/L or 1.5 ng/mL). Kinetics were rapid and cell-free studies showed that DT390-mIL-3 was as toxic as native DT. DT390-mIL-3 was selective because anti-mIL-3 monoclonal antibody, but not irrelevant antibody, inhibited its ability to kill. Cell lines not expressing IL-3R were not inhibited by the fusion protein. Because the use of DT390-mIL-3 as an antileukemia agent could be restricted by its reactivity with committed and/or primitive progenitor cells, bone marrow (BM) progenitor assays were performed. DT390-mIL-3 selectively inhibited committed BM progenitor cells as measured by in vitro colony-forming unit-granulocyte-macrophage and in vivo colony-forming unit-spleen colony assays. To determine if this fusion protein was reactive against BM progenitor cells required to rescue lethally irradiated recipients, adoptive transfer experiments were performed. Eight million DT390-mIL-3-treated C57BL/6 Ly5.2 BM cells, but not 4 million, were able to rescue lethally irradiated congenic C57BL/6 Ly5.1 recipients, suggesting that progenitor cells might be heterogenous in their expression of IL-3R. This idea was supported in competitive repopulation experiments in which DT390-mIL-3 treated C57BL/6 Ly5.2 BM cells were mixed with nontreated C57BL/6 Ly5.1 BM cells and used to reconstitute C57BL/6 Ly5.1 mice. A significant reduction

  3. 77 FR 4227 - Implantation or Injectable Dosage Form New Animal Drugs; Gonadotropin Releasing Factor Analog...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-27

    ... HUMAN SERVICES Food and Drug Administration 21 CFR Part 522 Implantation or Injectable Dosage Form New... gonadotropin releasing factor analog-diphtheria toxoid conjugate injectable solution. DATES: This rule is...: PART 522--IMPLANTATION OR INJECTABLE DOSAGE FORM NEW ANIMAL DRUGS 0 1. The authority citation for...

  4. Tetanus, Diphtheria, and Pertussis Vaccines

    MedlinePlus

    ... nose and throat. Whooping cough causes uncontrollable coughing. Vaccines can protect you from these diseases. In the U.S., there are four combination vaccines: DTaP prevents all three diseases. It is for ...

  5. Tetanus, Diphtheria, Pertussis (Tdap) Vaccine

    MedlinePlus

    ... the throat. It can lead to breathing problems, paralysis, heart failure, and death.PERTUSSIS (Whooping Cough) causes severe coughing spells, which can cause difficulty breathing, vomiting and disturbed sleep. It can also lead to weight loss, incontinence, ...

  6. Bypass of carrier-induced epitope-specific suppression using a T-helper epitope.

    PubMed Central

    Sad, S; Rao, K; Arora, R; Talwar, G P; Raghupathy, R

    1992-01-01

    A gonadotropin-releasing hormone (GnRH)-based vaccine is being developed as a method for non-surgical immunotherapy as immunization with this vaccine results in atrophy of the prostate. This vaccine, a conjugate of GnRH and diphtheria toxoid (DT), provides a unique hapten-carrier system for investigating the influence of carrier presensitization on antibody responses to self haptens. In a recent communication we showed that preimmunization with carriers diphtheria toxoid and tetanus toxoid results in a strain-dependent inhibition of anti-GnRH responses in mice and that T cells from carrier-presensitized mice are responsible for anti-haptenic suppression. In the present report we describe a strategy for bypassing DT-induced epitopic suppression using a T-helper epitope from DT. PMID:1383134

  7. 9 CFR 113.114 - Tetanus Toxoid.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... determined in antitoxin units (A.U.) per ml using an enzyme-linked immunosorbent assay method acceptable to... enzyme-linked immunosorbent assay. If at least 80 percent of the individual serums have an...

  8. 9 CFR 113.114 - Tetanus Toxoid.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... tested for potency. A group of 10 guinea pigs consisting of an equal number of males and females weighing... product labels. (1) Six weeks after injection, all surviving guinea pigs shall be bled and equal portions... serums have an antitoxin titer of at least 2.0 A.U. per ml, the serial may be retested in 10 guinea...

  9. 9 CFR 113.114 - Tetanus Toxoid.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... tested for potency. A group of 10 guinea pigs consisting of an equal number of males and females weighing... product labels. (1) Six weeks after injection, all surviving guinea pigs shall be bled and equal portions... serums have an antitoxin titer of at least 2.0 A.U. per ml, the serial may be retested in 10 guinea...

  10. Is diphtheria-tetanus-pertussis (DTP) associated with increased female mortality? A meta-analysis testing the hypotheses of sex-differential non-specific effects of DTP vaccine

    PubMed Central

    Aaby, Peter; Ravn, Henrik; Fisker, Ane B.; Rodrigues, Amabelia; Benn, Christine S.

    2016-01-01

    Background Ten years ago, we formulated two hypotheses about whole-cell diphtheria-tetanus-pertussis (DTP) vaccination: first, when given after BCG, DTP increases mortality in girls and, second, following DTP there is an increase in the female/male mortality rate ratio (MRR). A recent review by WHO found no convincing evidence that DTP increases mortality in females. Methods We used previous DTP reviews as well as the recent WHO review for assessing the hypotheses. As pre-specified we excluded studies with survival or frailty bias; if children had received BCG and DTP simultaneously; and if the children had received neonatal vitamin A. Results In seven studies of BCG-vaccinated children, DTP vaccination was associated with a 2.54 (95% CI 1.68–3.86) increase in mortality in girls (with no increase in boys [ratio 0.96, 0.55–1.68]). In 10 studies of BCG-vaccinated children, the female-to-male mortality ratio was 2.45 (1.48–4.06) times higher after DTP than before DTP. In 15 studies of children who had received DTP after previous BCG vaccination, mortality was 1.53 (1.21–1.93) times higher in girls than boys. The findings were similar in studies conducted before and after formulation of the hypotheses. Conclusions The two hypotheses were confirmed in the studies that fulfilled pre-specified criteria. PMID:27856947

  11. 9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... mixed with one-tenth unit of Standard Antitoxin and not cause sickness or death in injected mice. (iii... Antitoxin and cause death in at least 80 percent of injected mice. (iv) Standard antitoxin. The Epsilon... temperature for 1 hour and hold in ice water until injections of mice can be made. (vi) Five Swiss white...

  12. 9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... mixed with one-tenth unit of Standard Antitoxin and not cause sickness or death in injected mice. (iii... Antitoxin and cause death in at least 80 percent of injected mice. (iv) Standard antitoxin. The Epsilon... temperature for 1 hour and hold in ice water until injections of mice can be made. (vi) Five Swiss white...

  13. 9 CFR 113.111 - Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... mixed with one unit of Standard Antitoxin and not cause sickness or death in injected mice. (iii) L... death in at least 80 percent of injected mice. (iv) Standard antitoxin. The Beta Antitoxin preparation... toxin-antitoxin mixtures at room temperature for 1 hour and hold in ice water until injections of...

  14. 9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... mixed with one-tenth unit of Standard Antitoxin and not cause sickness or death in injected mice. (iii... Antitoxin and cause death in at least 80 percent of injected mice. (iv) Standard antitoxin. The Epsilon... temperature for 1 hour and hold in ice water until injections of mice can be made. (vi) Five Swiss white...

  15. 9 CFR 113.111 - Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... mixed with one unit of Standard Antitoxin and not cause sickness or death in injected mice. (iii) L... death in at least 80 percent of injected mice. (iv) Standard antitoxin. The Beta Antitoxin preparation... toxin-antitoxin mixtures at room temperature for 1 hour and hold in ice water until injections of...

  16. 9 CFR 113.111 - Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... mixed with one unit of Standard Antitoxin and not cause sickness or death in injected mice. (iii) L... death in at least 80 percent of injected mice. (iv) Standard antitoxin. The Beta Antitoxin preparation... toxin-antitoxin mixtures at room temperature for 1 hour and hold in ice water until injections of...

  17. 9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... mixed with one-tenth unit of Standard Antitoxin and not cause sickness or death in injected mice. (iii... Antitoxin and cause death in at least 80 percent of injected mice. (iv) Standard antitoxin. The Epsilon... temperature for 1 hour and hold in ice water until injections of mice can be made. (vi) Five Swiss white...

  18. 9 CFR 113.111 - Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... mixed with one unit of Standard Antitoxin and not cause sickness or death in injected mice. (iii) L... death in at least 80 percent of injected mice. (iv) Standard antitoxin. The Beta Antitoxin preparation... toxin-antitoxin mixtures at room temperature for 1 hour and hold in ice water until injections of...

  19. 9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... mixed with one-tenth unit of Standard Antitoxin and not cause sickness or death in injected mice. (iii... Antitoxin and cause death in at least 80 percent of injected mice. (iv) Standard antitoxin. The Epsilon... temperature for 1 hour and hold in ice water until injections of mice can be made. (vi) Five Swiss white...

  20. 9 CFR 113.111 - Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... mixed with one unit of Standard Antitoxin and not cause sickness or death in injected mice. (iii) L... death in at least 80 percent of injected mice. (iv) Standard antitoxin. The Beta Antitoxin preparation... toxin-antitoxin mixtures at room temperature for 1 hour and hold in ice water until injections of...

  1. Immunogenicity, safety, and antibody persistence at 3, 5, and 10 years postvaccination in adolescents randomized to booster immunization with a combined tetanus, diphtheria, 5-component acellular pertussis, and inactivated poliomyelitis vaccine administered with a hepatitis B virus vaccine concurrently or 1 month apart.

    PubMed

    Embree, Joanne; Law, Barbara; Voloshen, Tim; Tomovici, Antigona

    2015-03-01

    An understanding of the antibody persistence elicited by a combined tetanus, diphtheria, 5-component acellular pertussis, and inactivated poliovirus vaccine (Tdap-IPV) after adolescent vaccination is important to optimize booster dosing intervals. Our objectives were to compare the safety and immunogenicity of Tdap-IPV coadministered with hepatitis B vaccine (HepB) and sequential administration and evaluate humoral immunity at 3, 5, and 10 years after Tdap-IPV vaccination in adolescents. This phase II randomized, controlled, and open-label study enrolled 280 11- to 14-year-old adolescents with up to 10 years postvaccination follow-up. Group 1 (n = 145) received Tdap-IPV, followed by a HepB dose 1 month later, and group 2 (n = 135) received both vaccines simultaneously. No consistent increases in solicited reactions or unsolicited adverse events occurred with coadministration. All vaccinees attained seroprotective antibody levels at ≥0.01 IU/ml for diphtheria and tetanus, at a ≥1:8 dilution for poliovirus (serotypes 1, 2, and 3), and ≥10 mIU/ml for hepatitis B at 1 month postvaccination. Clinically relevant immunologic interactions did not occur with coadministration. For pertussis, all participants achieved seropositivity levels (at or above the lower limit of quantitation), and 72.7% to 95.8% had 4-fold increases in pertussis antibodies at 1 month postvaccination. At 10 years postvaccination, the remaining participants (62.8% of the original cohort) maintained seroprotective levels of ≥0.01 IU/ml for diphtheria and tetanus, a ≥1:8 dilution for all 3 poliovirus serotypes, and 74.1% to 98.2% maintained pertussis seropositivity levels depending on the antigen tested. There were no differences between the groups. These results support the coadministration of Tdap-IPV and HepB to adolescents and suggest that vaccination with Tdap-IPV can offer protection for 10 years after an adolescent booster vaccination.

  2. Immunogenicity, Safety, and Antibody Persistence at 3, 5, and 10 Years Postvaccination in Adolescents Randomized to Booster Immunization with a Combined Tetanus, Diphtheria, 5-Component Acellular Pertussis, and Inactivated Poliomyelitis Vaccine Administered with a Hepatitis B Virus Vaccine Concurrently or 1 Month Apart

    PubMed Central

    Embree, Joanne; Law, Barbara; Voloshen, Tim

    2014-01-01

    An understanding of the antibody persistence elicited by a combined tetanus, diphtheria, 5-component acellular pertussis, and inactivated poliovirus vaccine (Tdap-IPV) after adolescent vaccination is important to optimize booster dosing intervals. Our objectives were to compare the safety and immunogenicity of Tdap-IPV coadministered with hepatitis B vaccine (HepB) and sequential administration and evaluate humoral immunity at 3, 5, and 10 years after Tdap-IPV vaccination in adolescents. This phase II randomized, controlled, and open-label study enrolled 280 11- to 14-year-old adolescents with up to 10 years postvaccination follow-up. Group 1 (n = 145) received Tdap-IPV, followed by a HepB dose 1 month later, and group 2 (n = 135) received both vaccines simultaneously. No consistent increases in solicited reactions or unsolicited adverse events occurred with coadministration. All vaccinees attained seroprotective antibody levels at ≥0.01 IU/ml for diphtheria and tetanus, at a ≥1:8 dilution for poliovirus (serotypes 1, 2, and 3), and ≥10 mIU/ml for hepatitis B at 1 month postvaccination. Clinically relevant immunologic interactions did not occur with coadministration. For pertussis, all participants achieved seropositivity levels (at or above the lower limit of quantitation), and 72.7% to 95.8% had 4-fold increases in pertussis antibodies at 1 month postvaccination. At 10 years postvaccination, the remaining participants (62.8% of the original cohort) maintained seroprotective levels of ≥0.01 IU/ml for diphtheria and tetanus, a ≥1:8 dilution for all 3 poliovirus serotypes, and 74.1% to 98.2% maintained pertussis seropositivity levels depending on the antigen tested. There were no differences between the groups. These results support the coadministration of Tdap-IPV and HepB to adolescents and suggest that vaccination with Tdap-IPV can offer protection for 10 years after an adolescent booster vaccination. PMID:25540274

  3. Structures of the substrate-free and product-bound forms of HmuO, a heme oxygenase from corynebacterium diphtheriae: x-ray crystallography and molecular dynamics investigation.

    PubMed

    Unno, Masaki; Ardèvol, Albert; Rovira, Carme; Ikeda-Saito, Masao

    2013-11-29

    Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe(3+)-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe(3+)-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe(3+)-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe(3+)-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.

  4. Depletion of Dendritic Cells Enhances Susceptibility to Cell-Free Infection of Human T Cell Leukemia Virus Type 1 in CD11c-Diphtheria Toxin Receptor Transgenic Mice

    PubMed Central

    Rahman, Saifur; Manuel, Sharrón L.; Khan, Zafar K.; Wigdahl, Brian; Acheampong, Edward; Tangy, Frederic; Jain, Pooja

    2010-01-01

    Human T cell leukemia virus type 1 (HTLV-1) is associated with two immunologically distinct diseases: HTLV-1–associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. The genesis of these diseases is believed to be associated with the route (mucosa versus blood) and mode (cell-free versus cell-associated) of primary infection as well as the modulation of dendritic cell (DC) functions. To explore the role of DCs during early HTLV-1 infection invivo, we used a chimeric HTLV-1 with a replaced envelope gene from Moloney murine leukemia virus to allow HTLV-1 to fuse with murine cells, which are generally not susceptible to infection with human retroviruses. We also used a CD11c-diphtheria toxin receptor transgenic mouse model system that permits conditional transient depletion of CD11c+ DCs. We infected these transgenic mice with HTLV-1 using both cell-free and cell-associated infection routes in the absence and presence of DCs. The ablation of DCs led to an enhanced susceptibility to infection with cell-free but not cell-associated HTLV-1 in both CD4 and non-CD4 fractions, as measured by the proviral load. Infection with cell-free virus in the absence of DCs was also found to have increased levels of Tax mRNA in the non-CD4 fraction. Moreover, depletion of DCs significantly dampened the cellular immune response (IFN-γ+CD8+ T cells) against both cell-free and cell-associated virus. These results uniquely differentiate the involvement of DCs in early cell-free versus late cell-associated infection of HTLV-1 and highlight a significant aspect of viral immunopathogenesis related to the progression of adult T cell leukemia and HTLV-1–associated myelopathy/tropical spastic paraparesis after the initial infection. PMID:20382884

  5. DT388-GM-CSF, a novel fusion toxin consisting of a truncated diphtheria toxin fused to human granulocyte-macrophage colony-stimulating factor, prolongs host survival in a SCID mouse model of acute myeloid leukemia.

    PubMed

    Hall, P D; Willingham, M C; Kreitman, R J; Frankel, A E

    1999-04-01

    Despite significant advances in the treatment of acute myeloid leukemia (AML), the majority of patients will succumb to drug-resistant AML. To overcome this resistance, we have developed a novel fusion toxin consisting of the catalytic and translocation subunits of diphtheria toxin (DT388) linked to human granulocyte-macrophage colony-stimulating factor (GM-CSF). In vitro, DT388-GM-CSF demonstrated significant activity against numerous AML cell lines and fresh AML blasts. To determine its in vivo efficacy, we developed an in vivo model of human AML in severe combined immunodeficiency (SCID) mice injected intravenously with 1 x 10(7) HL-60 cells (AML-M2 cell line). The SCID mice developed abdominal masses, infiltration of the liver and bone marrow, and peripheral blasts with a median survival of 42.5 days. We tested DT388-GM-CSF, ara-C, human GM-CSF, and DAB389IL-2, which were injected intraperitoneally on days 2-6 in this model. DT3-GM-CSF significantly improved survival of the SCID mice over Ara-C, DAB389IL-2, or control (P < 0.001). DT388-GM-CSF-treated mice who developed leukemia exhibited no difference in the number of GM-CSF receptors (P = 0.39), ligand affinity (P = 0.77), or sensitivity (P = 0.56) to DT388-GM-CSF as compared to the controls. Frank leukemia in DT388-GM-CSF-treated mice may be due to incomplete penetration of drug into tissues rather than cellular resistance. DT388-GM-CSF is an active therapeutic agent in our SCID mouse model of AML with a unique mechanism of action and differing toxicities than current cytotoxic agents.

  6. Safety, immunogenicity and persistence of immune response to the combined diphtheria, tetanus, acellular pertussis, poliovirus and Haemophilus influenzae type b conjugate vaccine (DTPa-IPV/Hib) administered in Chinese infants

    PubMed Central

    Li, Yanping; Li, Rong Cheng; Ye, Qiang; Li, Changgui; Liu, You Ping; Ma, Xiao; Li, Yanan; Zhao, Hong; Chen, Xiaoling; Assudani, Deepak; Karkada, Naveen; Han, Htay Htay; Van Der Meeren, Olivier; Mesaros, Narcisa

    2017-01-01

    ABSTRACT We conducted 3 phase III, randomized, open-label, clinical trials assessing the safety, reactogenicity (all studies), immunogenicity (Primary vaccination study) and persistence of immune responses (Booster study) to the combined diphtheria, tetanus, pertussis, poliomyelitis, and Haemophilus influenzae type b vaccine (DTPa-IPV/Hib) in Chinese infants and toddlers. In the Pilot study (NCT00964028), 50 infants (randomized 1:1) received 3 doses of DTPa-IPV/Hib at 2–3–4 (Group A) or 3–4–5 months of age (Group B). In the Primary study (NCT01086423), 984 healthy infants (randomized 1:1:1) received 3 doses of DTPa-IPV/Hib at 2–3–4 (Group A) or 3–4–5 (Group B) months of age, or concomitant DTPa/Hib and poliomyelitis (IPV) vaccination at 2–3–4 months of age (Control group); 825 infants received a booster dose of DTPa/Hib and IPV at 18–24 months of age (Booster study; NCT01449812). In the Pilot study, unsolicited symptoms were more frequent in Group A (16 versus 1 infant; mostly upper respiratory tract infection and pyrexia); this observation was attributed to an epidemic outbreak of viral infections. Non-inferiority of 3-dose primary vaccination with DTPa-IPV/Hib over separately administered DTPa/Hib and IPV was demonstrated for Group A (primary objective). Similar antibody concentrations were observed in all groups, except for anti-polyribosyl-ribitol phosphate and anti-poliovirus types 1–3 which were higher in DTPa-IPV/Hib recipients. Protective antibody levels against all vaccine antigens remained high until booster vaccination. Three-dose vaccination with DTPa-IPV/Hib had a clinically acceptable safety profile. PMID:27768515

  7. Safety, immunogenicity and persistence of immune response to the combined diphtheria, tetanus, acellular pertussis, poliovirus and Haemophilus influenzae type b conjugate vaccine (DTPa-IPV/Hib) administered in Chinese infants.

    PubMed

    Li, Yanping; Li, Rong Cheng; Ye, Qiang; Li, Changgui; Liu, You Ping; Ma, Xiao; Li, Yanan; Zhao, Hong; Chen, Xiaoling; Assudani, Deepak; Karkada, Naveen; Han, Htay Htay; Van Der Meeren, Olivier; Mesaros, Narcisa

    2017-03-04

    We conducted 3 phase III, randomized, open-label, clinical trials assessing the safety, reactogenicity (all studies), immunogenicity (Primary vaccination study) and persistence of immune responses (Booster study) to the combined diphtheria, tetanus, pertussis, poliomyelitis, and Haemophilus influenzae type b vaccine (DTPa-IPV/Hib) in Chinese infants and toddlers. In the Pilot study (NCT00964028), 50 infants (randomized 1:1) received 3 doses of DTPa-IPV/Hib at 2-3-4 (Group A) or 3-4-5 months of age (Group B). In the Primary study (NCT01086423), 984 healthy infants (randomized 1:1:1) received 3 doses of DTPa-IPV/Hib at 2-3-4 (Group A) or 3-4-5 (Group B) months of age, or concomitant DTPa/Hib and poliomyelitis (IPV) vaccination at 2-3-4 months of age (Control group); 825 infants received a booster dose of DTPa/Hib and IPV at 18-24 months of age (Booster study; NCT01449812). In the Pilot study, unsolicited symptoms were more frequent in Group A (16 versus 1 infant; mostly upper respiratory tract infection and pyrexia); this observation was attributed to an epidemic outbreak of viral infections. Non-inferiority of 3-dose primary vaccination with DTPa-IPV/Hib over separately administered DTPa/Hib and IPV was demonstrated for Group A (primary objective). Similar antibody concentrations were observed in all groups, except for anti-polyribosyl-ribitol phosphate and anti-poliovirus types 1-3 which were higher in DTPa-IPV/Hib recipients. Protective antibody levels against all vaccine antigens remained high until booster vaccination. Three-dose vaccination with DTPa-IPV/Hib had a clinically acceptable safety profile.

  8. Are there altered antibody responses to measles, mumps, or rubella viruses in autism?

    PubMed

    Libbey, Jane E; Coon, Hilary H; Kirkman, Nikki J; Sweeten, Thayne L; Miller, Judith N; Lainhart, Janet E; McMahon, William M; Fujinami, Robert S

    2007-06-01

    The role that virus infections play in autism is not known. Others have reported that antibodies against measles virus are higher in the sera/plasma of children with autism versus controls. The authors investigated antibody titers to measles, mumps, and rubella viruses and diphtheria toxoid in children with autism, both classic onset (33) and regressive onset (26) forms, controls (25, healthy age- and gender-matched) and individuals with Tourette's syndrome (24) via enzyme-linked immunosorbent assays. No significant differences in antibody titers to measles, mumps, and rubella viruses and diphtheria toxoid were found among the four groups. Additionally, there were no significant differences between the four groups for total immunoglobulin (Ig)G or IgM. Interestingly, the authors did find a significant number (15/59) of autism subjects (classic and regressive onset combined) who had a very low or no antibody titer against rubella virus, compared to a combine control/Tourette's group (2/49).

  9. Interactions of conjugate vaccines and co-administered vaccines

    PubMed Central

    Findlow, H; Borrow, R

    2016-01-01

    Conjugate vaccines play an important role in the prevention of infectious diseases such as those caused by the bacteria Haemophilus influenzae (Hi) type b (Hib), Neisseria meningitidis, and Streptococcus pneumoniae. Vaccines developed against these 3 pathogens utilize 3 main carrier proteins, non-toxic mutant of diphtheria toxin (CRM197), diphtheria toxoid (DT) and tetanus toxoid (TT). Current pediatric immunisation schedules include the administration of several vaccines simultaneously, therefore increasing the potential for immune interference (both positively and negatively) to the antigens administered. Knowledge of vaccine interactions is principally derived from clinical trials, these are reviewed here to explore immune interference which may result of from carrier-specific T-cell helper interactions, bystander interference and carrier induced epitopic suppression. PMID:26619353

  10. The Evaluation of Jet Injection for use in Veterinary Medicine,

    DTIC Science & Technology

    1976-05-01

    Army "in-house" product testing commenced. A final scientific report was forwarded to Headquarters, Department of the Army, Surgeon General--Research and...included the first use of jet injection for administration of immunojens (diphtheria and tetanus toxoids). The authors found that toxin neutralizing...implicated jet injection as the cause of a fatal staphy - lococcal pneumonia in a child who received a routine 38 measles vaccination by jet injector

  11. Alginate Nanoparticles as a Promising Adjuvant and Vaccine Delivery System

    PubMed Central

    Sarei, F.; Dounighi, N. Mohammadpour; Zolfagharian, H.; Khaki, P.; Bidhendi, S. Moradi

    2013-01-01

    During last decades, diphtheria has remained as a serious disease that still outbreaks and can occur worldwide. Recently, new vaccine delivery systems have been developed by using the biodegradable and biocompatible polymers such as alginate. Alginate nanoparticles as a carrier with adjuvant and prolong release properties that enhance the immunogenicity of vaccines. In this study diphtheria toxoid loaded nanoparticles were prepared by ionic gelation technique and characterized with respect to size, zeta potential, morphology, encapsulation efficiency, release profile, and immunogenicity. Appropriate parameters (calcium chloride and sodium alginate concentration, homogenization rate and homogenization time) redounded to the formation of suitable nanoparticles with a mean diameter of 70±0.5 nm. The loading studies of the nanoparticles resulted in high loading capacities (>90%) and subsequent release studies showed prolong profile. The stability and antigenicity of toxoid were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and ouchterlony test and proved that the encapsulation process did not affect the antigenic integrity and activity. Guinea pigs immunized with the diphtheria toxoid-loaded alginate nanoparticles showed highest humoral immune response than conventional vaccine. It is concluded that, with regard to the desirable properties of nanoparticles and high immunogenicity, alginate nanoparticles could be considered as a new promising vaccine delivery and adjuvant system. PMID:24302799

  12. Trans-generational and neonatal humoral immune responses in West Greenland sledge dogs (Canis familiaris) exposed to organohalogenated environmental contaminants.

    PubMed

    Sonne, Christian; Larsen, Hans Jørgen S; Kirkegaard, Maja; Letcher, Robert J; Dietz, Rune

    2010-11-01

    Previous investigations in the Arctic have suggested OHC (organohalogen contaminant) induced immune toxic effects on e.g. polar bears (Ursus maritimus). We therefore studied the dietary impact from minke whale blubber (Balaenoptera acutorostrata), rich in polyunsaturated fatty acids and OHCs, on the humoral immunity of 7 captive West Greenland sledge dog (Canis familiaris) bitches and their 4 pups constituting a sentinel model species for polar bears. A control group was composed of 8 bitches and their 5 pups all fed pork (Suis scrofa) fat. The study included serum IgG measurements (bitches and pups) and specific immune responses towards tetanus toxoid (bitches) and diphtheria toxoid (pups) as well as influenza virus (pups). The analyses showed that IgG concentrations were non-significantly lowest in exposed bitches and pups (t-test: all p>0.05). In addition, significant lower antibody response was detected in exposed pups immunized with influenza virus at age 3 months (t-test: both p<0.05). No clear group differences were found for tetanus toxoid in bitches and diphtheria toxoid in pups. The results suggest that the humoral immune system of sledge dogs may be suppressed by the dietary blubber composition of OHCs and polyunsaturated fatty acids while a larger follow-up study is recommended in order to investigate this relationship further.

  13. Diphtheria, Tetanus, Pertussis: Ask the Experts

    MedlinePlus

    ... and then had a positive pertussis PCR two weeks later, could it be a false positive from ... during each pregnancy, preferably between 27 and 36 weeks' gestation. Women who have never received Tdap and ...

  14. Tdap (tetanus, diphtheria, pertussis) Vaccine and Pregnancy

    MedlinePlus

    ... as many of the antibodies as possible. These antibodies should provide some protection against pertussis before the baby can receive his/her own vaccines. If the mother, father, household members and other adult care givers ...

  15. Diphtheria, Tetanus, Pertussis: Ask the Experts

    MedlinePlus

    ... Td vaccine less than two years ago? Yes. Parents should receive a single dose of Tdap as soon as possible to protect their baby from pertussis. If a dose of Td was given within the previous 2 years, parents should still be vaccinated with Tdap as soon ...

  16. 9 CFR 113.108 - Clostridium Novyi Bacterin-Toxoid.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Alpha toxin-neutralization test provided in this paragraph. (1) When used in this test, the following... determined by the serum neutralization test as follows: (i) Make a dilution of Standard Antitoxin to contain..., the results of the serum neutralization test are inconclusive and shall be repeated: Provided,...

  17. 9 CFR 113.109 - Clostridium Sordellii Bacterin-Toxoid.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... potency using the toxin-neutralization test provided in this paragraph. (1) When used in this test, the... antitoxin content of the rabbit serums shall be determined by the serum neutralization test as follows: (i....0 Lo doses of Standard Toxin die, the results of the serum neutralization test are inconclusive...

  18. 9 CFR 113.108 - Clostridium Novyi Bacterin-Toxoid.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Alpha toxin-neutralization test provided in this paragraph. (1) When used in this test, the following... determined by the serum neutralization test as follows: (i) Make a dilution of Standard Antitoxin to contain..., the results of the serum neutralization test are inconclusive and shall be repeated: Provided,...

  19. 9 CFR 113.108 - Clostridium Novyi Bacterin-Toxoid.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Alpha toxin-neutralization test provided in this paragraph. (1) When used in this test, the following... determined by the serum neutralization test as follows: (i) Make a dilution of Standard Antitoxin to contain..., the results of the serum neutralization test are inconclusive and shall be repeated: Provided,...

  20. 9 CFR 113.109 - Clostridium Sordellii Bacterin-Toxoid.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... potency using the toxin-neutralization test provided in this paragraph. (1) When used in this test, the... antitoxin content of the rabbit serums shall be determined by the serum neutralization test as follows: (i....0 Lo doses of Standard Toxin die, the results of the serum neutralization test are inconclusive...

  1. 9 CFR 113.109 - Clostridium Sordellii Bacterin-Toxoid.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... potency using the toxin-neutralization test provided in this paragraph. (1) When used in this test, the... antitoxin content of the rabbit serums shall be determined by the serum neutralization test as follows: (i....0 Lo doses of Standard Toxin die, the results of the serum neutralization test are inconclusive...

  2. 9 CFR 113.108 - Clostridium Novyi Bacterin-Toxoid.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Alpha toxin-neutralization test provided in this paragraph. (1) When used in this test, the following... determined by the serum neutralization test as follows: (i) Make a dilution of Standard Antitoxin to contain..., the results of the serum neutralization test are inconclusive and shall be repeated: Provided,...

  3. 9 CFR 113.109 - Clostridium Sordellii Bacterin-Toxoid.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... potency using the toxin-neutralization test provided in this paragraph. (1) When used in this test, the... antitoxin content of the rabbit serums shall be determined by the serum neutralization test as follows: (i....0 Lo doses of Standard Toxin die, the results of the serum neutralization test are inconclusive...

  4. 9 CFR 113.109 - Clostridium Sordellii Bacterin-Toxoid.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Inspection Service. (vi) Diluent. The solution used to make proper dilutions prescribed in this test. Such solutions shall be made by dissolving 1 gram of peptone and 0.25 gram of sodium chloride in each 100 ml of....0 International Unit of Standard Antitoxin with 1.0 L+ dose of diluted Standard Toxin. Each...

  5. 9 CFR 113.108 - Clostridium Novyi Bacterin-Toxoid.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Animal and Plant Health Inspection Service. (vi) Diluent. The solution used to make proper dilutions prescribed in this test. Such solutions shall be made by dissolving 1 gram of peptone and 0.25 gram of sodium... Antitoxin with 0.1 L+ dose of diluted Standard Toxin. Each mixture is adjusted to a final volume of 2.0...

  6. 9 CFR 113.115 - Staphylococcus Aureus Bacterin-Toxoid.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    .... 113.115 Section 113.115 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... standard antitoxin produces a 50 percent hemolysis of rabbit red blood cells. (6) Incubate toxin-antitoxin... drawn rabbit red blood cells suspended in normal saline to each tube. Mix and incubate the...

  7. 9 CFR 113.115 - Staphylococcus Aureus Bacterin-Toxoid.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    .... 113.115 Section 113.115 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... standard antitoxin produces a 50 percent hemolysis of rabbit red blood cells. (6) Incubate toxin-antitoxin... drawn rabbit red blood cells suspended in normal saline to each tube. Mix and incubate the...

  8. Effect of vaccination with carrier protein on response to meningococcal C conjugate vaccines and value of different immunoassays as predictors of protection.

    PubMed

    Burrage, Moya; Robinson, Andrew; Borrow, Ray; Andrews, Nick; Southern, Joanna; Findlow, Jamie; Martin, Sarah; Thornton, Carol; Goldblatt, David; Corbel, Michael; Sesardic, Dorothea; Cartwight, Keith; Richmond, Peter; Miller, Elizabeth

    2002-09-01

    In order to plan for the wide-scale introduction of meningococcal C conjugate (MCC) vaccine for United Kingdom children up to 18 years old, phase II trials were undertaken to investigate whether there was any interaction between MCC vaccines conjugated to tetanus toxoid (TT) or a derivative of diphtheria toxin (CRM(197)) and diphtheria-tetanus vaccines given for boosting at school entry or leaving. Children (n = 1,766) received a diphtheria-tetanus booster either 1 month before, 1 month after, or concurrently with one of three MCC vaccines conjugated to CRM(197) or TT. All of the MCC vaccines induced high antibody responses to the serogroup C polysaccharide that were indicative of protection. The immune response to the MCC-TT vaccine was reduced as a result of prior immunization with a tetanus-containing vaccine, but antibody levels were still well above the lower threshold for protection. Prior or simultaneous administration of a diphtheria-containing vaccine did not affect the response to MCC-CRM(197) vaccines. The immune responses to the carrier proteins were similar to those induced by a comparable dose of diphtheria or tetanus vaccine. The results also demonstrate that, for these conjugate vaccines in these age groups, both standard enzyme-linked immunosorbent assays and those that measure high-avidity antibodies to meningococcal C polysaccharide correlated equally well with assays that measure serum bactericidal antibodies, the established serological correlate of protection for MCC vaccines.

  9. A randomized, dose-ranging assessment of the immunogenicity and safety of a booster dose of a combined diphtheria-tetanus-whole cell pertussis-hepatitis B-inactivated poliovirus-Hemophilus influenzae type b (DTPw-HBV-IPV/Hib) vaccine vs. co-administration of DTPw-HBV/Hib and IPV vaccines in 12 to 24 months old Filipino toddlers

    PubMed Central

    Quiambao, Beatriz; Van Der Meeren, Olivier; Kolhe, Devayani; Gatchalian, Salvacion

    2012-01-01

    As progress toward global poliovirus eradication continues, more and more countries are moving away from use of oral poliovirus vaccines (OPV) to inactivated poliovirus vaccines (IPV) in national vaccination schedules. Reduction of antigen dose in IPV could increase manufacturing capacity and facilitate the change from OPV to IPV. Combination vaccines reduce the number of injections required to complete vaccination, thus playing an important role in maintaining high vaccine coverage with good public acceptability. Three formulations of a combined, candidate hexavalent diphtheria-tetanus-whole cell pertussis-hepatitis B-inactivated poliovirus-Hemophilus influenzae type b conjugate vaccine (DTPw-HBV-IPV/Hib, GlaxoSmithKline Biologicals) differing only in IPV antigen content (full-dose, half-dose and one-third dose as compared with available stand-alone IPV vaccines), were evaluated when administered to healthy toddlers. Controls received separately administered licensed DTPw-HBV/Hib and IPV vaccines. Immunogenicity was assessed before and one month after vaccination. Safety and reactogenicity data were assessed for 30 d after vaccination. A total of 312 Filipino children were vaccinated in their second year of life. Each DTPw-HBV-IPV/Hib formulation was non-inferior to control in terms of pre-defined criteria for IPV immunogenicity. Post-vaccination GMTs against each poliovirus type were increased between 4.2- and 37.9-fold over pre-vaccination titers. Non-inferiority to other vaccine antigens was also demonstrated. The safety profile of the 3 DTPw-HBV-IPV/Hib formulations resembled licensed DTPw-HBV/Hib Kft and IPV in terms of the frequency and intensity of adverse reactions after vaccination. Further investigation of DTPw-HBV-IPV/Hib containing reduced quantity of IPV antigen for primary vaccination in infants is warranted.   This study is registered at www.clinicaltrials.gov NCT number: NCT01106092 PMID:22330958

  10. A randomized, dose-ranging assessment of the immunogenicity and safety of a booster dose of a combined diphtheria-tetanus-whole cell pertussis-hepatitis B-inactivated poliovirus-Hemophilus influenzae type b (DTPw-HBV-IPV/Hib) vaccine vs. co-administration of DTPw-HBV/Hib and IPV vaccines in 12 to 24 months old Filipino toddlers.

    PubMed

    Quiambao, Beatriz; Van Der Meeren, Olivier; Kolhe, Devayani; Gatchalian, Salvacion

    2012-03-01

    As progress toward global poliovirus eradication continues, more and more countries are moving away from use of oral poliovirus vaccines (OPV) to inactivated poliovirus vaccines (IPV) in national vaccination schedules. Reduction of antigen dose in IPV could increase manufacturing capacity and facilitate the change from OPV to IPV. Combination vaccines reduce the number of injections required to complete vaccination, thus playing an important role in maintaining high vaccine coverage with good public acceptability. Three formulations of a combined, candidate hexavalent diphtheria-tetanus-whole cell pertussis-hepatitis B-inactivated poliovirus-Hemophilus influenzae type b conjugate vaccine (DTPw-HBV-IPV/Hib, GlaxoSmithKline Biologicals) differing only in IPV antigen content (full-dose, half-dose and one-third dose as compared with available stand-alone IPV vaccines), were evaluated when administered to healthy toddlers. Controls received separately administered licensed DTPw-HBV/Hib and IPV vaccines. Immunogenicity was assessed before and one month after vaccination. Safety and reactogenicity data were assessed for 30 d after vaccination. A total of 312 Filipino children were vaccinated in their second year of life. Each DTPw-HBV-IPV/Hib formulation was non-inferior to control in terms of pre-defined criteria for IPV immunogenicity. Post-vaccination GMTs against each poliovirus type were increased between 4.2- and 37.9-fold over pre-vaccination titers. Non-inferiority to other vaccine antigens was also demonstrated. The safety profile of the 3 DTPw-HBV-IPV/Hib formulations resembled licensed DTPw-HBV/Hib Kft and IPV in terms of the frequency and intensity of adverse reactions after vaccination. Further investigation of DTPw-HBV-IPV/Hib containing reduced quantity of IPV antigen for primary vaccination in infants is warranted. This study is registered at www.clinicaltrials.gov NCT number: NCT01106092.

  11. Maternal immunization. Clinical experiences, challenges, and opportunities in vaccine acceptance.

    PubMed

    Moniz, Michelle H; Beigi, Richard H

    2014-01-01

    Maternal immunization holds tremendous promise to improve maternal and neonatal health for a number of infectious conditions. The unique susceptibilities of pregnant women to infectious conditions, as well as the ability of maternally-derived antibody to offer vital neonatal protection (via placental transfer), together have produced the recent increased attention on maternal immunization. The Advisory Committee on Immunization Practices (ACIP) currently recommends 2 immunizations for all pregnant women lacking contraindication, inactivated Influenza and tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (Tdap). Given ongoing research the number of vaccines recommended during pregnancy is likely to increase. Thus, achieving high vaccination coverage of pregnant women for all recommended immunizations is a key public health enterprise. This review will focus on the present state of vaccine acceptance in pregnancy, with attention to currently identified barriers and determinants of vaccine acceptance. Additionally, opportunities for improvement will be considered.

  12. Synthetic trimer and tetramer of 3-beta-D-ribose-(1-1)-D-ribitol-5-phosphate conjugated to protein induce antibody responses to Haemophilus influenzae type b capsular polysaccharide in mice and monkeys.

    PubMed Central

    Peeters, C C; Evenberg, D; Hoogerhout, P; Käyhty, H; Saarinen, L; van Boeckel, C A; van der Marel, G A; van Boom, J H; Poolman, J T

    1992-01-01

    Synthetic oligosaccharides derived from the capsular polysaccharide (PRP) of Haemophilus influenzae type b were conjugated to carrier proteins via a thioether linkage. Conjugates were made of trimeric and tetrameric ribose-ribitol-phosphate and tetanus toxoid or diphtheria toxin. All conjugates elicited anti-PRP antibody responses with an increasing immunoglobulin G/immunoglobulin M ratio in adult mice and monkeys. Trimer conjugates elicited lower anti-PRP antibody responses compared with tetramer conjugates. Adult monkeys responded equally well to the tetrameric oligosaccharide-tetanus toxoid conjugate as to the oligosaccharide-CRM197 conjugate (HbOC), which elicits protective levels of serum antibodies in human infants after two or three injections. PMID:1563770

  13. Vaccines and pregnancy: past, present, and future.

    PubMed

    Rasmussen, Sonja A; Watson, Amelia K; Kennedy, Erin D; Broder, Karen R; Jamieson, Denise J

    2014-06-01

    Vaccination during pregnancy with certain vaccines can prevent morbidity and mortality in pregnant women and their infants. However, previous recommendations often focused on the potential risks of vaccines to the fetus when used during pregnancy. In recent years, additional data have become available on the absence of increased risks for adverse events associated with vaccines when administered during pregnancy and on their benefits to mothers and infants. Currently two vaccines - (i) inactivated influenza, and (ii) tetanus toxoid, reduced diphtheria toxoid and acellular pertussis (Tdap) - are recommended for use by all pregnant women by the United States Advisory Committee on Immunization Practices. Here we review the history of vaccination during pregnancy, the current status of recommendations for vaccination during pregnancy in the USA, and the potential for future advances in this area, including key barriers that must be overcome to accommodate these advances.

  14. The preteen visit: an opportunity for prevention.

    PubMed

    Campos-Outcalt, Doug

    2006-12-01

    All early adolescents should visit a physician at age 11 or 12 years to receive a set of recommended vaccines. Two vaccines are recommended for boys in this age group-quadrivalent meningococcal conjugate vaccine (MCV4) and tetanus toxoid, reduced diphtheria, and acellular pertussis vaccine (Tdap). Three vaccines are recommended for girls--MCV4, Tdap, and human papilloma virus (HPV) vaccine. In addition, 2 doses of varicella vaccine are now recommended before age 5 years; both boys and girls at age 11 or 12 who have received only 1 dose should be given a second.

  15. Active Immunization—Some Present-Day Problems

    PubMed Central

    1941-01-01

    Diphtheria.—Immunization is safe and effective. Compulsory measures are indicated, especially for the younger age-groups. The Schick test may be reserved for selected groups and to control modified methods. Proper spacing of doses of prophylactics and periodic reinoculation will ensure a high level of immunity. It is important to beware of “one-shot” methods. Indiscriminate swabbing is to be discouraged; high carrier rates are an indication for widespread diphtheria prophylaxis. Enteric fever.—Mass immunization is desirable in many areas, although there is no justification for compulsion except for people exposed to special risks. In deciding upon dosage of vaccine, more attention should be paid to physical state and body-weight. After the primary course, very small periodic doses (for example o.i.c.c) are worthy of trial. Vaccine can be given during an epidemic without increasing the chances of infection. Tetanus.—Two doses of toxoid spaced by six weeks give useful immunity. Women give significantly higher titres than men. A third dose of i.o.c.c. after a long interval—seven to nine months—often produces a dramatic rise in circulating antitoxin, and counteracts any tendency to waning immunity. Smallpox.—As vaccination has not been made compulsory in this country, infection by virulent strains from the continent may tax the resources of the public health services. Whooping-cough.—The large number of injections of vaccine usually recommended is a deterrent to mass immunization. Research into the possibility of fewer doses and wider spacing is indicated. Other diseases are considered briefly. Combined immunization.—It may be useful to combine diphtheria T.A.F. and tetanus toxoid, also tetanus toxoid and T.A.B. vaccine. T.A.F. plus T.A.B. is probably contra-indicated for adults on account of severe reactions. Diphtheria A.P.T. should not be mixed with tetanus toxoid as it may go into solution and become ineffective. Sterilization of syringes and

  16. Td (tetanus and diphtheria) vaccine - what you need to know

    MedlinePlus

    ... Vaccine Information Statement (VIS): www.cdc.gov/vaccines/hcp/vis/vis-statements/td.pdf CDC review information ... Disease Control and website. www.cdc.gov/vaccines/hcp/vis/vis-statements/td.pdf . Accessed April 21, ...

  17. Long-term antibody memory induced by synthetic peptide vaccination is protective against Streptococcus pyogenes infection and is independent of memory T cell help.

    PubMed

    Pandey, Manisha; Wykes, Michelle N; Hartas, Jon; Good, Michael F; Batzloff, Michael R

    2013-03-15

    Streptococcus pyogenes (group A Streptococcus [GAS]) is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Vaccination with J8, a conserved region synthetic peptide derived from the M-protein of GAS and containing only 12 aa from GAS, when conjugated to diphtheria toxoid, has been shown to protect mice against a lethal GAS challenge. Protection has been previously shown to be Ab-mediated. J8 does not contain a dominant GAS-specific T cell epitope. The current study examined long-term Ab memory and dissected the role of B and T cells. Our results demonstrated that vaccination generates specific memory B cells (MBC) and long-lasting Ab responses. The MBC response can be activated following boost with Ag or limiting numbers of whole bacteria. We further show that these memory responses protect against systemic infection with GAS. T cell help is required for activation of MBC but can be provided by naive T cells responding directly to GAS at the time of infection. Thus, individuals whose T cells do not recognize the short synthetic peptide in the vaccine will be able to generate a protective and rapid memory Ab response at the time of infection. These studies significantly strengthen previous findings, which showed that protection by the J8-diphtheria toxoid vaccine is Ab-mediated and suggest that in vaccine design for other organisms the source of T cell help for Ab responses need not be limited to sequences from the organism itself.

  18. Antibody responses of Macaca fascicularis against a new inactivated polio vaccine derived from Sabin strains (sIPV) in DTaP-sIPV vaccine.

    PubMed

    Sato, Y; Shiosaki, K; Goto, Y; Sonoda, K; Kino, Y

    2013-05-01

    Antibody responses of Macaca fascicularis against a new tetravalent vaccine composed of diphtheria toxoid, tetanus toxoid, acellular pertussis antigens, and inactivated poliovirus derived from Sabin strains (sIPV) was investigated to predict an optimal dose of sIPV in a new tetravalent vaccine (DTaP-sIPV) prior to conducting a dose-defined clinical study. Monkeys were inoculated with DTaP-sIPVs containing three different antigen units of sIPVs: Vaccine A (types 1:2:3 = 3:100:100 DU), Vaccine B (types 1:2:3 = 1.5:50:50 DU), and Vaccine C (types 1:2:3 = 0.75:25:25 DU). There was no difference in the average titers of neutralizing antibody against the attenuated or virulent polioviruses between Vaccines A and B. The average neutralizing antibody titers of Vaccine C tended to be lower than those of Vaccines A and B. The sIPV antigens did not affect the anti-diphtheria or anti-tetanus antibody titers of DTaP-sIPV. Furthermore, the average neutralizing antibody titers of Vaccine A against the attenuated and virulent polioviruses were comparable between M. fascicularis and humans. These results suggest that M. fascicularis may be a useful animal model for predicting the antibody responses to sIPVs in humans, and that it may be likely to reduce the amount of sIPVs contained in DTaP-sIPVs, even for humans.

  19. Spotlight on DTPa-HBV-IPV/Hib Vaccine (Infanrix hexa).

    PubMed

    Dhillon, Sohita

    2010-10-01

    Infanrix hexa, administered intramuscularly, is a diphtheria, tetanus, acellular pertussis, hepatitis B (HBV), inactivated poliomyelitis and Haemophilus influenzae type b (Hib) conjugate vaccine, indicated for primary and booster vaccination of infants. Infanrix hexa should be administered as a two- or three-dose primary vaccination course in infants aged < or =6 months, followed by booster vaccination between 11 and 18 months of age, with an interval of at least 6 months between the last dose of primary vaccination and the booster dose. This spotlight reviews the immunogenicity and protective effectiveness, as well as the reactogenicity and safety of Infanrix hexa. Infanrix hexa as primary and booster vac