Llama heavy-chain antibody fragments efficiently remove toxic shock syndrome toxin 1 from plasma in vitro but not in experimental porcine septic shock.

Staphylococcus aureus produces the superantigen toxic shock syndrome toxin 1 (TSST-1). When the bacterium invades the human circulation, this toxin can induce life-threatening gram-positive sepsis. Current sepsis treatment does not remove bacterial toxins. Variable domains of llama heavy-chain antibodies (VHH) against toxic shock syndrome toxin 1 ([alpha]-TSST-1 VHH) were previously found to be effective in vitro. We hypothesized that removing TSST-1 with [alpha]-TSST-1 VHH hemofiltration filters would ameliorate experimental sepsis in pigs. After assessing in vitro whether timely removing TSST-1 interrupted TSST-1-induced mononuclear cell TNF-[alpha] production, VHH-coated filters were applied in a porcine sepsis model. Clinical course, survival, plasma interferon [gamma], and TSST-1 levels were similar with and without VHH-coated filters as were TSST-1 concentrations before and after the VHH filter. Plasma TSST-1 levels were much lower than anticipated from the distribution of the amount of infused TSST-1, suggesting compartmentalization to space or adhesion to surface not accessible to hemofiltration or pheresis techniques. Removing TSST-1 from plasma was feasible in vitro. However, the [alpha]-TSST-1 VHH adsorption filter-based technique was ineffective in vivo, indicating that improvement of VHH-based hemofiltration is required. Sequestration likely prevented the adequate removal of TSST-1. The latter warrants further investigation of TSST-1 distribution and clearance in vivo.

Specific immuno capturing of the staphylococcal superantigen toxic-shock syndrome toxin-1 in plasma.

Toxic-shock syndrome is primarily caused by the Toxic-shock syndrome toxin 1 (TSST-1), which is secreted by the Gram-positive bacterium Staphylococcus aureus. The toxin belongs to a family of superantigens (SAgs) which exhibit several shared biological properties, including the induction of massive cytokine release and V(beta)-specific T-cell proliferation. In this study we explored the possibility to use monoclonal Variable domains of Llama Heavy-chain antibodies (VHH) in the immuno capturing of TSST-1 from plasma. Data is presented that the selected VHHs are highly specific for TSST-1 and can be efficiently produced in large amounts in yeast. In view of affinity chromatography, the VHHs are easily coupled to beads, and are able to deplete TSST-1 from plasma at very low, for example, pathologically relevant, concentrations. When spiked with 4 ng/mL TSST-1 more than 96% of TSST-1 was depleted from pig plasma. These data pave the way to further explore application of high-affinity columns in the specific immuno depletion of SAgs in experimental sepsis models and in sepsis in humans.

Improved anti-IgG and HSA affinity ligands: clinical application of VHH antibody technology.

Large scale, highly specific purification of valuable proteins from blood and removal of undesirable components promise to have wide therapeutic applications. Moreover, depletion of bulk proteins from blood is a prerequisite for clinical proteomics. Here we describe the development of specific, high affinity Camelid antibody fragments (VHH) derived from immune libraries for purification and depletion of the bulk protein HSA and IgG from human serum and plasma for therapeutic and research purposes. The anti-IgG VHH substantially improved depletion of IgGs from blood over the classical method based on protein A. To demonstrate the improved performance of VHH based IgG depletion, we analyzed the presence of auto-antibodies in human plasma before and after depletion from two groups of patients with auto-immune disease: Goodpasture syndrome (GP) and systemic lupus erythematosus (SLE). VHHs can be produced efficiently and cost effectively in Saccharomyces cerevisiae, a genetically regarded as safe (GRAS) microorganism. A good manufacturing process (GMP) for purification of these VHHs has also been developed. Moreover, as VHHs are single protein chains, they can be coupled relatively easily to solid matrices. These three factors are important for developing affinity purification medication.

Llama single-chain antibody that blocks lipopolysaccharide binding and signaling: prospects for therapeutic applications.

Sepsis is a considerable health problem and a burden on the health care system. Endotoxin, or lipopolysaccharide (LPS), present in the outer membrane of gram-negative bacteria, is responsible for more than 50% of the sepsis cases and is, therefore, a legitimate target for therapeutic approaches against sepsis. In this study, we selected and characterized a llama single-chain antibody fragment (VHH) directed to Neisseria meningitidis LPS. The VHH, designated VHH 5G, showed affinity to purified LPS as well as to LPS on the surfaces of the bacteria. Epitope mapping using a panel of N. meningitidis mutants revealed that VHH 5G recognizes an epitope in the inner core of LPS, and as expected, the VHH proved to have broad specificity for LPS from different bacteria. Furthermore, this VHH blocked binding of LPS to target cells of the immune system, resulting in the inhibition of LPS signaling in whole blood. Moreover, it was found to remove LPS efficiently from aqueous solutions, including serum. The selected anti-LPS VHH is a leading candidate for therapies against LPS-mediated sepsis.

Efficient generation and amplification of high-capacity adeno-associated virus/adenovirus hybrid vectors.

Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in trans both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/loxP-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.