Michel Thiry

Lipases from psychrotropic antarctic bacteria

Properties of lipases excreted by 4 psychrotropic Moraxella strains from antarctic sea water have investigated. Despite an optimal cell generation time at 25°C, maximal lipase excretion essentially occurs at low temperatures. These enzymes are characterized by a shift of the apparent optimal activity towards low temperatures, by a reduction of the activation energy value and by a decrease in heat stability. These lipases are associated with lipopolysaccharides, forming high molecular weight complexes. One of the selected strain is able to express the antibiotic resistances carried by RP4, at both 4 and 25°C.

Cloning and Expression in Escherichia Coli of Three Lipase-Encoding Genes from the Psychrotrophic Antarctic Strain Moraxella Ta144

The cloning and expression of genes from a psychrotrophic bacterium in a mesophilic host are described. Three lipase (Lip)-encoding genes (lip) from the antarctic psychrotroph, Moraxella TA144, were cloned by inserting Sau3AI-generated DNA fragments into the BamHI site of the pSP73 plasmid vector. To prevent heat denaturation of the gene product, the screening procedure on agar plates containing an emulsified lipid involved growing of Escherichia coli recombinant colonies at 25 degrees C followed by incubation at 0 degree C. The three recombinant (reLip) were cell-associated and differed by their respective specificity towards p-nitrophenyl esters of various aliphatic chain lengths. These cloned reLip conserved the main character of the wild-type enzymes, i.e. a dramatic shift of the optimal temperature of activity towards low temperatures and pronounced heat lability.

Optimizing scale-up fermentation processes

There are many aims associated with the optimization of fermentation processes. Optimization is expected to increase the yield of the final product but the process must be compliant with good manufacturing practices, the available equipment and the expected final scale of operation. Dealing with genetically modified microorganisms that overproduce recombinant protein has the advantage that the vast majority of the processes use only three different species, namely Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. Standard processes for each organism are described in textbooks and serve as a basis for the development of a tailored process. This article outlines the general philosophy that we have devised to ensure an efficient approach of scaling up fermentation processes for biopharmaceutical purposes, in a multidisciplinary environment.

Safety and Immunogenicity of rSh28GST Antigen in Humans: Phase 1 Randomized Clinical Study of a Vaccine Candidate against Urinary Schistosomiasis

Background Treatment of urinary schistosomiasis by chemotherapy remains challenging due to rapid re-infection and possibly to limited susceptibility to praziquantel treatment. Therefore, therapeutic vaccines represent an attractive alternative control strategy. The objectives of this study were to assess the safety and tolerability profile of the recombinant 28 kDa glutathione S-transferase of Schistosoma haematobium (rSh28GST) in healthy volunteers, and to determine its immunogenicity. Methodology Volunteers randomly received 100 µg rSh28GST together with aluminium hydroxide (Alum) as adjuvant (n = 8), or Alum alone as a comparator (n = 8), twice with a 28-day interval between doses. A third dose of rSh28GST or Alum alone was administered to this group at day 150. In view of the results obtained, another group of healthy volunteers (n = 8) received two doses of 300 µg of rSh28GST, again with a 28-day interval. A six-month follow-up was performed with both clinical and biological evaluations. Immunogenicity of the vaccine candidate was evaluated in terms of specific antibody production, the capacity of sera to inhibit enzymatic activity of the antigen, and in vitro cytokine production. Principal Findings Among the 24 healthy male participants no serious adverse events were reported in the days or weeks after administration. Four subjects under rSh28GST reported mild reactions at the injection site while a clinically insignificant increase in bilirubin was observed in 8/24 subjects. No hematological nor biochemical evidence of toxicity was detected. Immunological analysis showed that rSh28GST was immunogenic. The induced Th2-type response was characterized by antibodies capable of inhibiting the enzymatic activity of rSh28GST. Conclusions rSh28GST in Alum did not induce any significant toxicity in healthy adults and generated a Th2-type immune response. Together with previous preclinical results, the data of safety, tolerability and quality of the specific immune response provide evidence that clinical trials with rSh28GST could be continued in humans as a potential vaccine candidate against urinary schistosomiasis.

A recombinant viral haemorrhagic septicaemia virus glycoprotein expressed in insect cells induces protective immunity in rainbow trout

Viral haemorrhagic septicaemia (VHS) is a fish rhabdovirus infection of world-wide importance. Control policies have been established but the disease still causes heavy losses in fish farming. The development of a recombinant subunit vaccine was initiated to produce a safe and effective vaccine to protect fish against VHS. The VHS virus (VHSV) glycoprotein, which induces neutralizing antibodies in rainbow trout, was chosen for expression in insect cells using a baculovirus vector. The M(r) of the recombinant protein estimated by SDS-PAGE was slightly lower than that of the native viral protein. The recombinant protein displayed different degrees of glycosylation and was recognized in ELISA by neutralizing antibodies. It was transported to the plasma membrane of insect cells where its ability to induce membrane fusion was preserved. The efficacy of the recombinant protein as a vaccine was compared with those of an inactivated and an attenuated vaccine. When injected intraperitoneally into rainbow trout, the baculovirus-encoded protein was shown (i) to induce the synthesis of VHSV-neutralizing antibodies and (ii) to confer protection against virus challenge. Immunization performed by immersion failed. This is the first report of a recombinant vaccine that protects fish against VHSV.

Cloning and sequencing the messenger RNA of the N gene of viral haemorrhagic septicaemia virus.

The mRNA transcribed from the N gene of viral haemorrhagic septicaemia virus (VHSV) of salmonids has been cloned in Escherichia coli and expressed. Fusion proteins were recognized by monoclonal antibody directed against the N protein from the viral particle. A 1212 bp long open reading frame (ORF) coding for 404 amino acids with a calculated Mr of 44590 was deduced from the nucleotide sequence. The ORF was preceded by a 93 bp segment including in position 42 the AACAC pentanucleotide which is presumed to be the start signal for transcription by analogy with other rhabdoviral mRNAs. The upstream 41 bp region could correspond to the covalently linked positive polarity leader RNA as also found on the N mRNA from infectious haematopoietic necrosis virus (IHNV). This may be a characteristic of fish lyssaviruses. The AAACC sequence, which is part of the leader, was not found. Amino acids 44 to 359 from IHNV and 45 to 360 from VHSV are 45.3% homologous. A strong homology which could reflect functional importance was also found for potential phosphorylation sites and hydrophobic peaks despite the fact that the two viruses evolved on different continents.

Nucleotide sequence of the lipase gene lip3 from the antarctic psychotroph Moraxella TA144

A lipase gene (lip3) from the psychotrophic strain Moraxella TA144 has been cloned and sequenced. The deduced primary structure of the lipase preprotein is composed of 315 amino acids with a predicted Mr of 34,772. This enzyme contains two consensus peptides showing cluster of glycine residues that may be involved in domain flexibility. The cloned gene product conserves the low temperature activity and the thermolability properties of the wild enzyme.