Vaccine manufacture at the time of a pandemic influenza.

In the event of a major influenza epidemic, the availability of a potent and safe vaccine would be a major concern. The following presentation describes the main features of a flu vaccine manufacturing campaign: beginning with the supply of embryonated eggs, in which the flu viruses are cultivated, through the different steps of vaccine production - egg harvest, purification, inactivation, splitting - down to the final vaccine formulation and aseptic filling in the appropriate containers. In usual times, such a production cycle takes over 70 weeks. In an emergency situation, the manufacturers and the authorities would have to take innovative approaches to minimize such delays. This will inevitably translate into an enormous strain on all the players in such a project, from the egg suppliers to the organisers of the vaccine dispatching and administration. It will result in suboptimal yields and costs. However, facing a massive and urgent need of vaccine, both the authorities and the vaccine manufacturers must work together to supply the necessary doses in time.

Improved distribution of antigenic site specificity of poliovirus-neutralizing antibodies induced by a protease-cleaved immunogen in mice.

Previous studies showed that the distribution of antigenic site specificity of neutralizing antibodies to type 3 poliovirus obtained with the inactivated poliovirus vaccine can be deficient as compared with that obtained following poliovirus infection. This observation was shown by the relatively low capacity of sera from inactivated-poliovirus-vaccine-immunized persons to neutralize poliovirus cleaved at antigenic site 1. We investigated possibilities for improving the situation in a mouse model. Balb/c mice were immunized with intact or trypsin-cleaved type 3 poliovirus (Saukett strain). Sera from mice immunized with the intact virus readily neutralized the intact virus but neutralized the cleaved virus only rarely. In contrast, cleaved-virus-immunized mice produced antibodies that were able to neutralize the cleaved virus as well as the intact one. Mice immunized with a 100-fold-higher dose of the intact virus produced significant levels of antibodies to the cleaved virus, too. Somewhat surprisingly, mice immunized with high doses of the cleaved virus produced antibodies specific for the intact loop between beta sheets B and C of VP1 (virion protein 1), which should be cleaved in the immunogen. This was shown by a higher titer of antibodies to intact Saukett virus than to the corresponding cleaved virus, as well as to a type 1/type 3 hybrid poliovirus in which only the BC loop amino acids were derived from type 3 poliovirus. The cleavage-induced enhanced availability of antigenic determinants residing outside the BC loop was also shown by increased neutralization titers of monoclonal antibodies specific for some of these other determinants. These results indicate that by using a trypsin-cleaved type 3 poliovirus as a parenteral immunogen, it is possible to change the distribution of antigenic site specificities of neutralizing antibodies to resemble that following poliovirus infection.

A new method for monitoring the sterility of blood donation.

Sterility of blood products is a cardinal contributor to patient safety. Bacteriologic controls of stable products comply with strict regulations, but legislation imposes only limited constraints in the case of perishable products, such as packed red cells (RBCs) or fresh-frozen plasma (FFP). Therefore, it is essential to monitor the sterility of aseptic donations from uninfected donors. Such bacteriologic monitoring can now be carried out through a tertiary bag (containing a soybean casein culture medium) connected to the classical double-pack system. This system does not jeopardize the sterility of the whole system, as the connection is tightly stoppered by a membrane. After the blood drawing, this tertiary bag is filled with 5 ml of blood, and separated from the rest of the system. It is then incubated for 3 days at 30 degrees C and for 14 days at 22 degrees C, to test for eventual bacteriologic or fungal contamination. In order to check the feasibility of this technique, we studied 76 blood drawings in the control laboratory of the blood center, and the results confirm the value of this system.

Chromosomal localization of gut, fruC, and pfk mutations affecting genes involved in Bacillus subtilis D-glucitol catabolism.

Mutations affecting the genes involved in B. subtilis D-glucitol catabolism were mapped either by PBS1-mediated transduction or DNA-mediated transformation. It was shown that the genes gutA and gutB coding for the D-glucitol permease and the D-glucitol dehydrogenase, respectively, and regulatory locus gutR are clustered in a gut operon localized between purB and dal close to the pha marker. A mutation affecting fructokinase activity (fruC) was mapped near the gut markers. The fruC gene does not belong to the operon. A mutation affecting phosphofructokinase activity (pfk) was mapped between the leuA and aroG markers.

Biochemical and genetic study of D-glucitol transport and catabolism in Bacillus subtilis.

The catabolic pathway of D-glucitol (sorbitol) in Bacillus subtilis Marburg 168M is characterized. It includes (i) a transport step catalyzed by a D-glucitol permease which is affected by the gutA mutations, (ii) an oxidation step of the intracellular D-glucitol catalyzed by a D-glucitol dehydrogenase, generating intracellular fructose, affected by gutB mutations, and (iii) phosphorylation of the intracellular fructose either at the C1 site or at the C6 site as described previously (A. Delobbe et al., Eur. J. Biochem., 66:485-491, 1976; A. Delobbe et al., EUR. J. Biochem. 51:503-510, 1975). Additional data are given concerning the phosphorylation of fructose by a fructokinase (fructose ATP 6-phosphotransferase), which is affected by the fruC mutation. The isolation of regulatory mutants affected in gutR that synthesize constitutively both the permease and the dehydrogenase indicates the existence of a D-glucitol operon in B. subtilis. Unlike the wild-type strain, these mutants are able to utilize D-xylitol as sole carbon source.

Phosphorylation of intracellular fructose in Bacillus subtilis mediated by phosphoenolpyruvate-1-fructose phosphotransferase.

Intracellular fructose provided by the sorbitol pathway in Bacillus subtilis can be phosphorylated by the phosphenolpyruvate-1-fructose phosphotransferase which is known to mediate a vectorial metabolism. The fate of this intracellular fructose was studied using mutants lacking either the fructose 1-phosphate pathway or the fructose 6-phosphate pathway. It was shown that the phosphoenolpyruvate-dependent phosphorylation needs a prior exit of the sugar into the medium, this exit being probably catalysed by a transport system. A low affinitiy intracellular phosphenolpyruvate phosphotransferase system was found, which seems to be devoid of a physiological role.

Existence of two alternative pathways for fructose and sorbitol metabolism in Bacillus subtilis Marburg.

Strains of Bacillus subtilis mutated for fructose phosphotransferase system (fruA), fructose-1-phosphate kinase (fruB), fructokinase (frucC) have been tested for their catabolism of sorbitol and fructose. It is shown that the previously known pathways of sorbitol and fructose degradation in B. subtilis, e.g.: (see article) may metabolize intracellular fructose produced either by sorbitol oxidation or by fructose-1-phosphate dephosphorylation. The intracellular fructore degradation via fructose-1-phosphate kinase has been found to require the fructose phosphotransferase system which ensures a vectorial transport of fructose.