Development of a single dose tetanus toxoid formulation based on polymeric microspheres: a comparative study of poly(D,L-lactic-co-glycolic acid) versus chitosan microspheres.

Stable polymeric microspheres capable of controlled release of tetanus toxoid (TT) for periods ranging from days to over months were developed. TT was stabilized, encapsulated in microspheres prepared from poly(D,L)-lactide-co-glycolide (PLGA) and chitosan by using protein stabilizer (trehalose) and its immune response was compared. The influence of co-encapsulated protein stabilizer on tetanus toxoid's stability and release from the microspheres was studied. The protein stabilizer (trehalose) prevented structural losses and aggregation of microencapsulated TT. To neutralize the acids liberated by the biodegradable lactic/glycolic acid-based polymer, we also co-incorporated into the polymer an antacid, (Mg(OH)2), which neutralized the acidity during degradation of the polymer and also prevented TT structural losses and aggregation. The in vitro release experiments with PLGA and chitosan microspheres were performed and the release of TT was increased up to 80-90%. The antigen integrity was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by coomassie brilliant blue staining. The SDS-PAGE analysis confirmed that antigen integrity was not affected by the encapsulation procedure. In addition, the immunogenicity of PLGA and chitosan microspheres based single dose vaccine was evaluated in guinea pigs and compared with multiple doses of alum adsorbed TT. Results indicated that a single injection of PLGA and chitosan microspheres containing TT could maintain the antibody response at a level comparable to the booster injections of conventional alum adsorbed vaccines. The both PLGA and chitosan based stable vaccine formulations produced an equal immune response. Hence chitosan can be used to replace the expensive polymer PLGA. This approach should have potential application in the field of vaccine delivery.

Process optimization for enhanced production of diphtheria toxin by submerged cultivation.

When stationary culture was replaced by submerged cultivation in a fermentor, a significant increase in the yield of diphtheria toxin in a short cultivation time (less than 48 h as against 7-8 d) was noted. It was found that under optimal conditions of temperature, vortex mixing and surface aeration, an alkaline pH favoured toxin release. Furthermore, to enhance the production volume, two-and three-step semicontinuous batch cultivations were carried out. The toxin produced was of good titre with an adequate antigenic purity. Under optimal conditions, the variation in the Limes of flocculation (Lf titre) was likely due to the quality of the production medium, which in turn depended on the quality of the raw materials used. The process was also optimized in a pilot-scale fermentor.

Preliminary report on a single-tube, non-interrupted reverse transcription-polymerase chain reaction for the detection of rabies virus in brain tissue.

A simple method for the rapid detection of rabies virus was developed employing a single-tube reverse transcription polymerase chain reaction (RT-PCR). The method utilized a single buffer system for both RT and PCR and was performed without interruption as a single thermal cycling programme. Two primer sets within the genes coding for rabies nucleoprotein and glycoprotein were used to amplify a 533 bp and a 406 bp amplicon, respectively. The amplified products were detected with a challenge virus strain (CVS) of rabies. There was no amplified product with uninfected mouse or dog brain. The method was applied to detect rabies virus in 10 mouse inoculation test (MIT)-positive and three MIT-negative brain tissue samples. The amplified product was found only in the MIT-positive samples. The amplified product was confirmed by restriction endonuclease analysis using Hinf1. The results from RT-PCR correlated well with the results from MIT. This indicates that the single-tube RT-PCR may be a useful method for detecting rabies virus in brain tissue samples from suspected cases of rabies.