Recombinant Antigens Expressed in Pichia pastoris for the Diagnosis of Sleeping Sickness Caused by Trypanosoma brucei gambiense

Background

Screening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.

Methodology/Principal Findings

We have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.

Conclusions/Significance

The diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.     

Evidence for the Involvement of the Small Subunit of HIV-1 Reverse Transcriptase (RT) in the TSAO-Resistance

Abstract

By introducing the TSAO-resistance mutation 138Glu→Lys in only the p51 subunit of HIV-1 RT, we obtained compelling evidence for a structural and functional role of the p51 subunit in the sensitivity and/or resistance of the enzyme to the TSAO-derivatives.     

Inversion and deletion mutants in Bacillus subtilis bacteriophage SPP1 as a consequence of cloning

Summary Properties of an inversion and a deletion mutant of B. subtilis phage SPP1 which arose during cloning are described. The results are related to the biology of this bacteriophage.In preceding communications from our laboratories (Heilmann and Reeve 1982, Behrens et al. 1983) we reported the properties of genetically engineered SPP1 bacteriophages, which could be used as cloning vehicles in B. subtilis. These phages contain a unique restriction site within a dispensable region of their genomes. In the course of cloning experiments using these phage vectors, we have occasionally observed the appearance of not only the original vector and desired hybrid phages, but also of SPP1 phages which had undergone extensive genomic rearrangements. Properties of two such phages, SPP1 inv1, which was found to contain a large inversion and of SPP1 delV, a deletion mutant, which defines an additional dispensable region of the SPP1 genome, are described in this communication.     

Comparative analysis of the immune responses induced by native versus recombinant versions of the ASP-based vaccine against the bovine intestinal parasite Cooperia oncophora

The protective capacities of a native double-domain activation-associated secreted protein (ndd-ASP)-based vaccine against the cattle intestinal nematode Cooperia oncophora has previously been demonstrated. However, protection analysis upon vaccination with a recombinantly produced antigen has never been performed. Therefore, the aim of the current study was to test the protective potential of a Pichia-produced double-domain ASP (pdd-ASP)-based vaccine against C. oncophora. Additionally, we aimed to compare the cellular and humoral mechanisms underlying the vaccine-induced responses by the native (ndd-ASP) and recombinant vaccines. Immunisation of cattle with the native C. oncophora vaccine conferred significant levels of protection after an experimental challenge infection, whereas the recombinant vaccine did not. Moreover, vaccination with ndd-ASP resulted in a higher proliferation of CD4-T cells both systemically and in the small intestinal mucosa when compared with animals vaccinated with the recombinant antigen. In terms of humoral response, although both native and recombinant vaccines induced similar levels of antibodies, animals vaccinated with the native vaccine were able to raise antibodies with greater specificity towards ndd-ASP in comparison with antibodies raised by vaccination with the recombinant vaccine, suggesting a differential immune recognition towards the ndd-ASP and pdd-ASP. Finally, the observation that animals displaying antibodies with higher percentages of recognition towards ndd-ASP also exhibited the lowest egg counts suggests a potential relationship between antibody specificity and protection.     

Complete nucleotide sequence of the new simian T-lymphotropic virus, STLV-PH969 from a Hamadryas baboon, and unusual features of its long terminal repeat.

A third type of primate T-lymphotropic virus, PTLV-L, with STLV-PH969 as a prototype, has recently been isolated from an African baboon (Papio hamadryas). Classification of this virus has been based on partial sequence analysis of cDNA from a virus-producing cell line, PH969. We obtained the complete nucleotide sequence of this virus with a proviral genome of 8,916 bp. All major genes, homologous in all human T-cell lymphotropic virus (HTLV)-related viruses, and their corresponding mRNAs, including appropriate splicing, were identified. One additional nonhomologous open reading frame in the proximal pX region is accessible for translation through alternative splicing. Sequence comparison shows that STLV-PH969 is equidistantly related to HTLV type 1 (HTLV-1) and HTLV-2. In all coding regions, the similarity tends to be the lowest between STLV-PH969 and HTLV-1. However, in the long terminal repeat (LTR) region, the lowest similarity was found between STLV-PH969 and HTLV-2. The U3-R and R-U5 boundaries of the STLV-PH969 LTR were experimentally determined at nucleotides 268 and 524, respectively. This 695-bp LTR is 60 and 73 bp shorter than the LTRs of HTLV-1 and HTLV-2, respectively, but its general organization is similar to the one found in the HTLV-bovine leukemia virus genus. In the long region between the polyadenylation signal and the poly(A) site, sequence similarity with the HTLV-1 Rex-responsive element (RexRE) core and secondary structure prediction suggest the presence of a RexRE. The presence of three 21-bp repeats is conserved within the U3 region of HTLV-1, HTLV-2, and BLV. Only two direct repeats with similarity to these Tax-responsive elements were found in the STLV-PH969 LTR, which might suggest differences in the Tax-mediated transactivation of this virus. We conclude that STLV-PH969 has all the genes and genomic regions to suggest a replication cycle comparable to that of HTLV-1 and HTLV-2.     

Characterization of a novel enzyme—<Emphasis Type="Italic">Starmerella bombicola</Emphasis> lactone esterase (SBLE)—responsible for sophorolipid lactonization

We recently discovered a novel enzyme in the exoproteome of Starmerella bombicola, which is structurally related to Candida antarctica lipase A. A knockout strain for this enzyme does no longer produce lactonic sophorolipids, prompting us to believe that this protein is the missing S. bombicola lactone esterase (SBLE). SBLE catalyzes a rather unusual reaction, i.e., an intramolecular esterification (lactonization) of acidic sophorolipids in an aqueous environment, which raised questions about its activity and mode of action. Here, we report the heterologous production of this enzyme in Pichia pastoris and its purification in a two-step strategy. Purified recombinant SBLE (rSBLE) was used to perform HPLC and liquid chromatography mass spectrometry (LCMS)-based assays with different sophorolipid mixtures. We experimentally confirmed that SBLE is able to perform ring closure of acetylated acidic sophorolipids. This substrate was selected for rSBLE kinetic studies to estimate the apparent values of K _m . We established that rSBLE displays optimal activity in the pH range of 3.5 to 6 and has an optimal temperature in the range of 20 to 50 °C. Additionally, we generated a rSBLE mutant through site-directed mutagenesis of Ser₁₉₄ in the predicted active site pocket and show that this mutant is lacking the ability to lactonize sophorolipids. We therefore propose that SBLE operates via the common serine hydrolase mechanism in which the catalytic serine residue is assisted by a His/Asp pair.     

An electron microscopic study of the structural polymorphism of hepatitis B antigen from human sera.

The physical features of hepatitis B antigen (HBAg) particles from human sera are investigated with electron microscopy and immune electron microscopic technique. All the virus-like particles are pleomorphic in structure; although they are classified into two categories: HBsAg and HBcAg by immunological technique. The small, spherical particles measured in a range of 16 to 30 nm in diameter, mostly 20 to 22 nm,are populous in the positive serum. The tubular particles have the width of 18 to 22 nm and the length of 50 to 230 nm or even longer. Sometimes these particles contain a larger end and become the tadpole shape. The large particles or Dane particles measured mainly 42 nm in diameter have an inner core and the outer coats. The inner core of 27 nm in diameter can expose spontaneously. It can be released from the coats by heating at 56 degrees C for 30 min or by treatment of Tween 80 (1%) at room temperature. When specific antibody is added in the positive sample, the aggregate of the antigen-antibody clumping can be revealed with electron microscope. The core antigen of the large particles may attach to the molecular protein of the antibody and show the spike-like structure. This polymorphism of HBAg particles seems unique in animal virology. The roles of these particles played in medical and virological fields are discussed.