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Background: The diphtheria toxoid antigen is a major component in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. Although antibodies are considered critical for diphtheria protection, little is known about the antigenic determinants that maintain humoral immunity. Methods: One-hundred and twelve 15 mer peptides covering the entire sequence of diphtheria toxin (DTx) protein were prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice immunized with a triple DTP vaccine allowed mapping of continuous B-cell epitopes, topological studies, multiantigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Results: Twenty epitopes were identified, with two being in the signal peptide, five in the catalytic domain (CD), seven in the HBFT domain, and five in the receptor-binding domain (RBD). Two 17 mer (CB/Tx-2/12 and CB/DTx-4-13) derived biepitope peptides linked by a Gly-Gly spacer were chemically synthesized. The peptides were used as antigens to coat ELISA plates and assayed with human (huVS) and mice vaccinated sera (miVS) for in vitro diagnosis of diphtheria. The assay proved to be highly sensitive (99.96%) and specific (100%) for huVS and miVS and, when compared with a commercial ELISA test, demonstrated a high performance. Conclusions: Our work displayed the complete picture of the linear B cell IgG response epitope of the DTx responsible for the protective effect and demonstrated sufficient specificity and eligibility for phase IIB studies of some epitopes to develop new and fast diagnostic assays.

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Diphtheria is a potentially fatal infection, mostly caused by diphtheria toxin (DT)-producing Corynebacterium diphtheriae strains. During the last decades, the isolation of DT-producing C. diphtheriae strains has been decreasing worldwide. However, non-DT-producing C. diphtheriae strains emerged as causative agents of cutaneous and invasive infections. Although endemic in countries with warm climates, cutaneous diphtheria is rarely reported in Brazil. Presently, an unusual case of skin lesion in a Brazilian elderly diabetic patient infected by a penicillin-resistant non-DT-producing C. diphtheriae strain was reported. Laboratory diagnosis included mass spectrometry and multiplex PCR analyses. Since cutaneous diphtheria lesions are possible sources of secondary diphtheria cases and systemic diseases and considering that penicillin is the first line of antimicrobial agent for the treatment of these infections, the detection of penicillin-resistant strains of diphtheria bacilli should be a matter of concern. Thus, cases similar to the presently reported should be appropriately investigated and treated, particularly in patients with risk factor (s) for the development of C. diphtheriae invasive infections, such as diabetes. Moreover, health professionals must be aware of the presence of C. diphtheriae in cutaneous lesions of lower limbs, a common type of morbidity in diabetic patients, especially in tropical and subtropical countries.

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Tetanus and diphtheria are diseases that still cause significant morbidity and mortality. Clostridium tetani produces the tetanus toxin, a 150-kDa protein. The diphtheria toxin is synthesized by Corynebacterium diphtheriae as a protein of 58 kDa. The objective of this study was to carry out a chemical characterization of the tetanus and diphtheria toxin forms in the several production process stages, and thus to establish an affordable alternative in vitro quality control to aggregate to the classical tests. The 150 kDa band of the tetanus toxin and approximately 58 kDa band of the diphtheria toxin were observed by electrophoresis similar as that described in the literature. The same band of 58 KDa was detected in Western blotting reactions. The results obtained for diphtheria toxin showed very similar protein profiles between distinct lots. For the tetanus toxin, the profiles of the initial stage showed some variability, but the ones of the following stages were similar. The similarity of the electrophoresis results indicated reproduction and consistency of the production processes in Butantan Institute and correlated with the yield and antigenic purity classical data. The establishment of alternative in vitro quality control tests can significantly contribute to achieve the consistency approach supported by WHO.

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Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR) assay that can simultaneously identify and determine the toxigenicity of these corynebacterial species with zoonotic potential was developed. This assay uses five primer pairs targeting the following genes: rpoB (Corynebacterium spp), 16S rRNA (C. ulcerans and C. pseudotuberculosis), pld (C. pseudotuberculosis), dtxR (C. diphtheriae) and tox [diphtheria toxin (DT) ]. In addition to describing this assay, we review the literature regarding the diseases caused by these pathogens. Of the 213 coryneform strains tested, the mPCR results for all toxigenic and non-toxigenic strains of C . diphtheriae, C. ulcerans and C. pseudotuberculosis were in 100% agreement with the results of standard biochemical tests and PCR-DT. As an alternative to conventional methods, due to its advantages of specificity and speed, the mPCR assay used in this study may successfully be applied for the diagnosis of human and/or animal diseases caused by potentially toxigenic corynebacterial species.

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We assessed the IgG levels anti-diphtheria (D-Ab) and T cell counts (CD4+ and CD8+) in HIV-1 infected subjects undergoing or not highly active antiretroviral therapy (HAART). Approximately 70% of all HIV-1 patients were unprotected against diphtheria. There were no differences in D-Ab according to CD4 counts. Untreated patients had higher D-Ab (geometric mean of 0.62 IU/ml) than HAART-patients (geometric mean of 0.39 IU/ml). The data indicated the necessity of keeping all HIV-1 patients up-to-date with their vaccination.

Assuntos

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We assessed the IgG levels anti-diphtheria (D-Ab) and T cell counts (CD4+ and CD8+) in HIV-1 infected subjects undergoing or not highly active antiretroviral therapy (HAART). Approximately 70% of all HIV-1 patients were unprotected against diphtheria. There were no differences in D-Ab according to CD4 counts. Untreated patients had higher D-Ab (geometric mean of 0.62 IU/ml) than HAART-patients (geometric mean of 0.39 IU/ml). The data indicated the necessity of keeping all HIV-1 patients up-to-date with their vaccination.

RESUMO

We assessed the IgG levels anti-diphtheria (D-Ab) and T cell counts (CD4+ and CD8+) in HIV-1 infected subjects undergoing or not highly active antiretroviral therapy (HAART). Approximately 70% of all HIV-1 patients were unprotected against diphtheria. There were no differences in D-Ab according to CD4 counts. Untreated patients had higher D-Ab (geometric mean of 0.62 IU/ml) than HAART-patients (geometric mean of 0.39 IU/ml). The data indicated the necessity of keeping all HIV-1 patients up-to-date with their vaccination.

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Se evaluó el uso de la tecnología de Flujo de Filtración Tangencial (FFT), para obtener la toxina diftérica a partir de cultivos de la bacteria Corynebacterium diphtheriae, usando el proceso de Microfiltración (MF), para eliminar el paquete celular y posteriormente, a partir del filtrado obtenido, concentrar y diafiltrar la toxina diftérica usando el proceso de Ultrafiltración (UF). Se determinaron características de los filtros, condiciones de trabajo y dimensionamiento de los equipos a adquirir para la producción industrial de Toxina Diftérica. Se evaluaron el flujo, tiempo, rendimiento del proceso y las características del producto obtenido, utilizando cultivos con Toxina Diftérica en un equipo de filtración de laboratorio, diseñado para producir el efecto de FFT. Seseleccionó las membranas tipo cassettes, formato Médium Screen, porosidad 0,2 μm, como las adecuadas para el proceso de MF, ya que mostraron 100% de transmisión de la Toxina Diftérica, ausencia de restos celulares y flujo promedio de filtrado de 9.16 L/m2h. Así mismo, se seleccionaron las membranas tipo cassettes, formato Omega, porosidad 10 y 30 kDa, como las adecuadas para el proceso de UF, ya que mostraron 100% de recuperación de la toxina, ausencia de toxina en el filtrado y adecuados flujos de filtrado (97,5 y 125,9 L/m2h, respectivamente), Estos resultaron permitieron dimensionar, considerando las variables a utilizar en la producción industrial (Volumen 650 a 950 Litros, Tiempo de Procesos, 3 a 5 horas), el área de filtración de los equipos de MF y UF a adquirir, estimados en 20m2 y 5m2, respectivamente.

Tangential Flow Filtration (TFF) technology was evaluated to process diphtheria toxin which is produced by Cory ne - bacterium diphtheriae bacterium. Microfiltration (MF) is used to retain cells while allowing passage of the toxin to the filtrate stream. The filtrate is collected and further pro - cessed by Ultrafiltration (UF) to concentrate the toxin and to maximize the wash of small species by a Diafiltration step. Both, MF and UF processes were evaluated to specify the filters and corresponding critical process parameters to scale-up the application. As part of the evaluation, flow rate, processing time, yield and product attributes were characterized. The cell harvest containing the diphtheria toxin was processed using a laboratory scale TFF system designed to product the TFF effect. The evaluation demonstrated that a cassette in medium screen format and membrane with 0.2 μm pore is the right selection for the MF step. It showed 100% of toxin transmission without the presence of cellular debris and average process flux of 9.16 L/m2h. The UF step was conducted using the same laboratory equipment with cassettes in medium screen format with pores of 10 and 30 kD. It showed 100% retention of the toxin with a process flux of 97,5 and 125,9 L/m2h, respectively. These results were used to scale-up the application to process the industrial volume of 650 a 950 liters between 3 to 5 hours of processing time. Membrane area sizing of MF and UF to be acquired is estimated in 20 m2 and 5m2, respectively.

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Infections caused by Corynebacterium diphtheriae frequently induce situations in which very small doses of antigens injected intradermally can cause strong inflammatory reactions. This bacterium secretes the diphtheria toxin (DT), a virulence factor that can be lethal to the human organism at doses below 0.1 mg/kg of body weight. The present work proposes alternative methods of DT purification using affinity chromatography and of DT detoxification through conjugating with the polymer methoxypolyethylene glycol activated(mPEG). Tests were performed to evaluate: the formation of edemas and the presence of dermonecrotic activity, in vitro cytotoxicity to Vero cells, the neutralizing activity of serum from guinea pigs immunized with the diphtheria toxoid inactivated with mPEG, and the immunogenic activity of the purified and modified toxin. The results indicated that purification with Blue Sepharose was an efficient method, yielding antigen purity equivalent to 2600 Lf/mg of protein nitrogen. The modification of the Purified Toxin with mPEG did not result in the formation of edema or necrosis although it was immunogenic and stimulated the formation of antibodies that could neutralize the Purified Toxin. The toxoidobtained from the purified toxin maintained its immunogenic characteristics, inducing antibodies with neutralizing activity; edema and necrosis were still observed, however.

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The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

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