An alternative method for purifying and detoxifying diphtheria toxin

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 mu g/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 toxoid obtained from the purified toxin maintained its immunogenic characteristics, inducing antibodies with neutralizing activity; edema and necrosis were still observed, however. (C) 2011 Elsevier Ltd. All rights reserved

Development of Equine IgG Antivenoms against Major Snake Groups in Mozambique.

BACKGROUND:Snake envenoming is a significant public health problem in underdeveloped and developing countries. In sub-Saharan Africa, it is estimated that 90,000-400,000 envenomations occur each year, resulting in 3,500-32,000 deaths. Envenomings are caused by snakes from the Viperidae (Bitis spp. and Echis spp.) and Elapidae (Naja spp. and Dendroaspis spp.) families. The African continent has been suffering from a severe antivenom crisis and current antivenom production is only sufficient to treat 25% of snakebite cases. Our aim is to develop high-quality antivenoms against the main snake species found in Mozambique. METHODS:Adult horses primed with the indicated venoms were divided into 5 groups (B. arietans; B. nasicornis + B. rhinoceros; N. melanoleuca; N. mossambica; N. annulifera + D. polylepis + D. angusticeps) and reimmunized two times for antivenom production. Blood was collected, and plasma was separated and subjected to antibody purification using caprylic acid. Plasmas and antivenoms were subject to titration, affinity determination, cross-recognition assays and in vivo venom lethality neutralization. A commercial anti-Crotalic antivenom was used for comparison. RESULTS:The purified antivenoms exhibited high titers against B. arietans, B. nasicornis and B. rhinoceros (5.18 x 106, 3.60 x 106 and 3.50 x 106 U-E/mL, respectively) and N. melanoleuca, N. mossambica and N. annulifera (7.41 x 106, 3.07 x 106 and 2.60 x 106 U-E/mL, respectively), but lower titers against the D. angusticeps and D. polylepis (1.87 x 106 and 1.67 x 106 U-E/mL). All the groups, except anti-N. melanoleuca, showed significant differences from the anti-Crotalic antivenom (7.55 x 106 U-E/mL). The affinity index of all the groups was high, ranging from 31% to 45%. Cross-recognition assays showed the recognition of proteins with similar molecular weight in the venoms and may indicate the possibility of paraspecific neutralization. The three monospecific antivenoms were able to provide in vivo protection. CONCLUSION:Our results indicate that the anti-Bitis and anti-Naja antivenoms developed would be useful for treating snakebite envenomations in Mozambique, although their effectiveness should to be increased. We propose instead the development of monospecific antivenoms, which would serve as the basis for two polyvalent antivenoms, the anti-Bitis and anti-Elapidae. Polyvalent antivenoms represent an increase in treatment quality, as they have a wider range of application and are easier to distribute and administer to snake envenoming victims

Artificial neural network associated to UV/Vis spectroscopy for monitoring bioreactions in biopharmaceutical processes

Currently, mammalian cells are the most utilized hosts for biopharmaceutical production. The culture media for these cell lines include commonly in their composition a pH indicator. Spectroscopic techniques are used for biopharmaceutical process monitoring, among them, UV–Vis spectroscopy has found scarce applications. This work aimed to define artificial neural networks architecture and fit its parameters to predict some nutrients and metabolites, as well as viable cell concentration based on UV–Vis spectral data of mammalian cell bioprocess using phenol red in culture medium. The BHK-21 cell line was used as a mammalian cell model. Off-line spectra of supernatant samples taken from batches performed at different dissolved oxygen concentrations in two bioreactor configurations and with two pH control strategies were used to define two artificial neural networks. According to absolute errors, glutamine (0.13 ± 0.14 mM), glutamate (0.02 ± 0.02 mM), glucose (1.11 ± 1.70 mM), lactate (0.84 ± 0.68 mM) and viable cell concentrations (1.89 105 ± 1.90 105 cell/mL) were suitably predicted. The prediction error averages for monitored variables were lower than those previously reported using different spectroscopic techniques in combination with partial least squares or artificial neural network. The present work allows for UV–VIS sensor development, and decreases cost related to nutrients and metabolite quantifications

Use of uniform designs in combination with neural networks for viral infection process development

This work aimed to compare the predictive capacity of empirical models, based on the uniform design utilization combined to artificial neural networks with respect to classical factorial designs in bioprocess, using as example the rabies virus replication in BHK-21 cells. The viral infection process parameters under study were temperature (34°C, 37°C), multiplicity of infection (0.04, 0.07, 0.1), times of infection, and harvest (24, 48, 72 hours) and the monitored output parameter was viral production. A multilevel factorial experimental design was performed for the study of this system. Fractions of this experimental approach (18, 24, 30, 36 and 42 runs), defined according uniform designs, were used as alternative for modelling through artificial neural network and thereafter an output variable optimization was carried out by means of genetic algorithm methodology. Model prediction capacities for all uniform design approaches under study were better than that found for classical factorial design approach. It was demonstrated that uniform design in combination with artificial neural network could be an efficient experimental approach for modelling complex bioprocess like viral production. For the present study case, 67% of experimental resources were saved when compared to a classical factorial design approach. In the near future, this strategy could replace the established factorial designs used in the bioprocess development activities performed within biopharmaceutical organizations because of the improvements gained in the economics of experimentation that do not sacrifice the quality of decisions

Electrophoretic profile and cross-recognition by antivenoms as assessed by western blotting (WB).

<p>(a) SDS-Page, upper gel 5%, lower gel 12.5%, stained with silver. (b) WB using anti-<i>B arietans</i> antivenom. (c) WB using anti-<i>B nasicornis + B</i>. <i>rhinoceros</i> antivenom. (d) WB using anti-<i>N</i>. <i>melanoleuca</i> antivenom. (e) WB using anti-<i>N</i>. <i>mossambica</i> antivenom. (f) WB using anti-<i>D</i>. <i>angusticeps + D</i>. <i>polylepis + N</i>. <i>annulifera</i> antivenom. In all experiments, 10 μg of crude venom was resolved under non-reducing conditions. The antibody dilution was fixed at 1:20,000.</p

Affinity of experimental and standard (anti-<i>Crotalic</i>) antivenoms after IgG purification.

<p>(a) Affinity curves and (b) affinity index comparison. The plates were sensitized with 1 μg of antigen/well and the plasma dilutions fixed at 1:20,000. The KSCN dilution ranged from 0 M to 5 M in 1 M intervals. The affinity was calculated as the percentage of antibodies bound at KSCN equal to 5 M; the dotted line represents 100%. The medians were compared using a one-way ANOVA followed by the Dunnett’s Multiple Comparison Test (standard antivenom as comparison), ***P < 0.05. The data represent three independent experiments.</p

Affinity of experimental plasmas (2^nd immunization) and standard anti-<i>Crotalic</i> antivenom before IgG purification.

<p>(a) Affinity curves and (b) affinity index comparison. The plates were sensitized with 1 μg of antigen/well, and the plasma dilutions were fixed at 1:20,000. The KSCN dilution ranged from 0 M to 5 M in 1 M intervals. The affinities were calculated as the percentage of antibodies bound at KSCN equal to 5 M; the dotted line represents 100%. The medians were compared by one-way ANOVA followed by the Dunnett’s Multiple Comparison Test (standard antivenom as comparison), *P < 0.05. The data represent three independent experiments.</p

Titers obtained from experimental antivenoms in cross-recognition assays after IgG purification.

<p>(a) anti-<i>B</i>. <i>arietans</i> antivenom. (b) anti-<i>B</i>. <i>nasicornis + B</i>. <i>rhinoceros</i> antivenom. (c) anti-<i>N</i>. <i>melanoleuca</i> antivenom. (d) anti-<i>N</i>. <i>mossambica</i> antivenom. (e) anti-<i>D</i>. <i>angusticeps + D</i>. <i>polylepis + N</i>. <i>annulifera</i> antivenom. The plates were sensitized with 1 μg of antigen/well and the plasma dilutions ranged from 1:2,000 to 1:1,024,000. The titers are expressed as Units-ELISA/mL x 10⁶. The data represent two independent experiments.</p

Titration from experimental plasmas and standard anti-<i>Crotalic</i> antivenom before IgG purification.

<p>(a)Titration curves and (b) titers comparison. The plates were sensitized with 1 μg of antigen/well and the plasma dilutions ranged from 1:4,000 to 1:512,000. The dotted line represents an OD of 0.2. The titers are expressed as Units-ELISA/mL x 10⁶. The medians were compared by two-way ANOVA followed by the Bonferroni Post-Test (standard antivenom as comparison), *P < 0.05, **P < 0.01, and ***P < 0.001. The data are representative of three independent experiments.</p