Process intensification for production of Streptococcus pneumoniae whole-cell vaccine.

The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a nonencapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for bacterial vaccine production.

Modeling Neurocognitive Decline and Recovery During Repeated Cycles of Extended Sleep and Chronic Sleep Deficiency.

Study Objectives: Intraindividual night-to-night sleep duration is often insufficient and variable. Here we report the effects of such chronic variable sleep deficiency on neurobehavioral performance and the ability of state-of-the-art models to predict these changes.

Methods: Eight healthy males (mean age ± SD: 23.

Polysaccharide purification from Haemophilus influenzae type b through tangential microfiltration.

Haemophilus influenzae type b (Hib) is a human pathogen that causes meningitis in infants worldwide. Capsular polysaccharide linked to a protein has been used as an efficient vaccine, and this approach has reduced the incidence of Hib disease since its inclusion in national immunisation campaigns. The traditional polysaccharide downstream process is based on several ethanol precipitations, treatment with detergents and centrifugation.

Development of a whole cell pneumococcal vaccine: BPL inactivation, cGMP production, and stability.

Pneumococcal infections impose a large burden of disease on the human population, mainly in developing countries, and the current pneumococcal vaccines offer serotype-specific protection, but do not cover all pathogenic strains, leaving populations vulnerable to disease caused by non-vaccine serotypes. The pneumococcal whole cell vaccine is a low-cost strategy based on non-capsular antigens common to all strains, inducing serotype-independent immunity. Therefore, we developed the process for the cGMP production of this cellular vaccine.

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 μg/kg of body weight.

GMP-grade pneumococcal whole-cell vaccine injected subcutaneously protects mice from nasopharyngeal colonization and fatal aspiration-sepsis.

Mucosal immunization with a killed whole-cell pneumococcal vaccine, given with enterotoxin-related adjuvants, has been shown to confer multi-serotype protection against colonization of the nasopharynx and middle ear in mice. However, because novel mucosal immunization strategies may be difficult to implement, here we evaluated subcutaneous injection. Strain RM200 was engineered to be capsule-negative, autolysin-negative, and to express a non-toxic mutant pneumolysoid.

Fed-batch production of tetanus toxin by Clostridium tetani.

This study deals with the effects of the initial nitrogen source (NZ Case TT) level and the protocol of glucose addition during the fed-batch production of tetanus toxin by Clostridium tetani. An increase in the initial concentration of NZ Case TT (NZ(0)) accelerated cell growth, increased the consumption of the nitrogen source as well as the final yield of tetanus toxin, which achieved the highest values (50-60 L(f)/mL) for NZ(0) > or = 50 g/L. The addition of glucose at fixed times (16, 56, and 88 h) ensured a toxin yield ( approximately 60 L(f)/mL) about 33% higher than those of fed-batch runs with addition at fixed concentration ( approximately 45 L(f)/mL) and about 300% higher than those obtained in reference batch runs nowadays used at industrial scale.

Enzymatic profiling of tetanus and botulinum neurotoxins based on vesicle-associated-membrane protein derived fluorogenic substrates.

Botulinum (BoNT) and tetanus (TeNT) neurotoxins are bacterial zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. There are seven serotypes of BoNT, while TeNT is found in one serotype. In order to characterize their enzymatic activities and to propose serotype-differentiation an enzymatic assay based on their metalloprotease activity was developed.

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

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.

Effect of medium composition on the production of tetanus toxin by Clostridium tetani.

The tetanus toxin is a neurotoxin synthesized by the bacillus Clostridium tetani that, after detoxification with formaldehyde, still exhibits antigenic and immunologic properties, hence its denomination of tetanus toxoid. Such a neurotoxin is produced by cultivation of the microorganism in vegetative form on a relatively complex specific medium containing glucose and peptone. The simultaneous effects of the starting levels of glucose (G0) and N-Z Case TT (NZ0) as carbon and nitrogen sources, respectively, on the production of tetanus toxin have been investigated in this work in static cultivations by means of a five-level star-shaped experimental design and evaluated by response surface methodology (RSM) for optimization purposes.

Development of an operational synaptobrevin-based fluorescent substrate for tetanus neurotoxin quantification.

Tetanus neurotoxin (TTx), produced by Clostridium tetani, is a two-chain polypeptide with a heavy molecular chain (HC; 100 kDa) and a light molecular chain (LC; 50 kDa) linked by a disulphide bridge. The low-molecular-mass chain is classified as a zinc metalloprotease (EC 3.4.

Use of sephacryl high resolution (HR) in tetanus anatoxin purification.

The tetanus purified anatoxin is used in the preparation of the tetanus toxoid and multiple vaccines (dT, DT and DTP), all of them strictly following specifications established by the WHO with a minimum antigenic purity equal to 1,000 Lf/mgPN. Aiming to establish more sensitive and accurate methods for purification, samples from four different lots of tetanus anatoxin were submitted to gel filtration in twenty independent trials using the Sephacryl S-100 HR and S-200 HR resins. The Authors were careful to optimize their parameters of performance as to sample volume, elution and selectivity flow for tetanus anatoxin purification, allowing their use in industrial scale.

Development and validation study for the chromatographic purification process for tetanus anatoxin on Sephacryl S-200 High Resolution.

The tetanus purified anatoxin is used in the preparation of multiple immunoprophylactics. WHO (World Health Organization) specifies that the tetanus anatoxin must exhibit a degree of purity greater than or equal to 1,000 Lf/mg protein nitrogen (PN). Today liquid chromatography is a well established technique for the purification of tetanus anatoxin and several different methods are used in production scale.

Specific peptides of casein pancreatic digestion enhance the production of tetanus toxin.

Casein pancreatic digest is the basic bacterial growth medium used for diphtheria, botulinum and tetanus toxin vaccine production. It is known that the variation in the peptide content of the casein digest directly affects final toxin yields. In this study, the identification and sequences of eight peptides, four to eight amino acids in length, of casein pancreatic digestion, which seem to be involved in the enhancement of tetanus toxin production, are described.