Synergistic Effect of Two Nanotechnologies Enhances the Protective Capacity of the Theileria parva Sporozoite p67C Antigen in Cattle.

East Coast fever (ECF), caused by Theileria parva, is the most important tick-borne disease of cattle in sub-Saharan Africa. Practical disadvantages associated with the currently used live-parasite vaccine could be overcome by subunit vaccines. An 80-aa polypeptide derived from the C-terminal portion of p67, a sporozoite surface Ag and target of neutralizing Abs, was the focus of the efforts on subunit vaccines against ECF and subjected to several vaccine trials with very promising results. However, the vaccination regimen was far from optimized, involving three inoculations of 450 µg of soluble p67C (s-p67C) Ag formulated in the Seppic adjuvant Montanide ISA 206 VG. Hence, an improved formulation of this polypeptide Ag is needed. In this study, we report on two nanotechnologies that enhance the bovine immune responses to p67C. Individually, HBcAg-p67C (chimeric hepatitis B core Ag virus-like particles displaying p67C) and silica vesicle (SV)-p67C (s-p67C adsorbed to SV-140-C18, octadecyl-modified SVs) adjuvanted with ISA 206 VG primed strong Ab and T cell responses to p67C in cattle, respectively. Coimmunization of cattle (Bos taurus) with HBcAg-p67C and SV-p67C resulted in stimulation of both high Ab titers and CD4 T cell response to p67C, leading to the highest subunit vaccine efficacy we have achieved to date with the p67C immunogen. These results offer the much-needed research depth on the innovative platforms for developing effective novel protein-based bovine vaccines to further the advancement.

Folding properties of the hepatitis B core as a carrier protein for vaccination research.

The hepatitis B core (HBc) protein has been used successfully in numerous experiments as a carrier for heterologous peptides. Folding and capsid formation of the chimeric proteins is not always achieved easily. In silico analyses were performed to provide further comprehension of the feasibility for predicting successful capsid formation. In contrast to previous work, we show that common in silico predictions do not ensure assembly into particles. We included new considerations regarding capsid formation of HBc fusion proteins. Not only the primary sequence and the length of the inserts seem important, also the rigidity, the distance between the N and the C-terminus and the presence of cysteines, which could form disulphide bonds, could influence proper capsid formation. Furthermore, new conformational insights were formulated when linkers were added to create extra flexibility of the chimeric particles. Different hypotheses were suggested to clarify the obtained results. To this extent, the addition of glycine-rich linkers could lower high rigidity of the insert, removal of the strain of the core protein or ease interaction between the HBc and the insert. Finally, we observed specific changes in capsid formation properties when longer linkers were used. These findings have not been reported before in this and other virus-like particle carriers. In this study, we also propose a new high-yield purification protocol for fusion proteins to be used in vaccination experiments with the carrier protein or in comparative studies of particulate or non-particulate HBc fusion proteins.

Transfection of live, tick derived sporozoites of the protozoan Apicomplexan parasite Theileria parva.

Theileria parva is an important veterinary protozoan causing the tick-borne disease East Coast fever. Transfection of Theileria parasites will facilitate the investigation of many aspects of this apicomplexan infection and its unique host-parasite interaction. The pathogen has the extraordinary capacity of transforming B and T cells into clonally dividing cancerous cell lines in a reversible way. Sequence data of the entire T. parva genome are available and in vitro infected cell lines can easily be generated, thereby eliminating the use of animals in the evaluation of the evolution of the transfected sporozoites. Here we report, for the first time, on experiments towards successful transient transfection of T. parva sporozoites, making use of a new generation transfection device. Plasmid DNA containing the strong EF-1α promoter and an Azami Green reporter gene were integrated by nucleofection into freshly purified T. parva sporozoites. Expression of Azami Green was detected with a fluorescence microscope and confirmed by counter staining with a monoclonal directed against a sporozoite protein. Despite the fact that transfection efficiencies are still low, this is the first step towards a stable infection method of T. parva parasites. In the long run, transfected parasites might become an alternative way to induce immunity without clinical signs.

Identification of a functional Antigen5-related allergen in the saliva of a blood feeding insect, the tsetse fly.

Our previous screening of a Glossina morsitans morsitans lamdagt11 salivary gland expression library with serum of a tsetse fly exposed rabbit identified a cDNA encoding Tsetse Antigen5 (TAg5, 28.9 kDa), a homologue of Antigen5 sting venom allergens. Recombinant TAg5 was produced in Sf9 cells in order to assess its immunogenic properties in humans. Plasma from a patient that previously exhibited anaphylactic reactions against tsetse fly bites contained circulating anti-TAg5 and anti-saliva IgEs. In a significant proportion of plasma samples of African individuals, TAg5 and saliva binding IgEs (respectively 56 and 65%) can be detected. Saliva, harvested from flies that were subjected to TAg5- specific RNA interference (RNAi), displayed significantly reduced IgE binding potential. Allergenic properties of TAg5 and tsetse fly saliva were further illustrated in immunized mice, using an immediate cutaneous hypersensitivity and passive cutaneous anaphylaxis assay. Collectively, TAg5 was illustrated to be a tsetse fly salivary allergen, demonstrating that Antigen5-related proteins are represented as functional allergens not only in stinging but also in blood feeding insects.

Comparison of serum humoral responses induced by oral immunization with the hepatitis B virus core antigen and the cholera toxin B subunit.

The hepatitis B virus core (HBc) virus-like particle (VLP) is known as one of the most immunogenic antigens and carrier vehicles in different immunization strategies. Recent findings are suggesting the potential of the HBc VLPs as an oral immunogen. Here, we focus on the induction of serum humoral responses by oral administration of HBc VLPs in preparations substantially free of lipopolysaccharide and immunomodulating encapsidated RNA. The full-length HBc antigen was used, because the C-terminal arginine-rich tail may contribute to the immunogenicity of the antigen as the region is involved in cell surface heparan sulfate binding and internalization of the protein. Serum antibody levels and isotypes were determined following oral administration of the HBc VLPs with the perspective of using the HBc VLP as an immunostimulatory and carrier molecule for epitopes of blood-borne diseases in oral immunization vaccination strategies. Following oral administration of the HBc VLP preparations to mice, a strong serum humoral response was induced with mainly immunoglobulin G2a (IgG2a) antibodies, pointing toward a Th1 response which is essential in the control of intracellular pathogens. Intraperitoneal immunization with the HBc VLP induced a stronger, mixed Th1/Th2 response. Finally, a comparison was made with the induced serum humoral response following oral administration of the recombinant cholera toxin B pentamer, a commonly used oral immunogen. These immunizations, in contrast, induced predominantly antibodies of the IgG1 isotype, indicative of a Th2 response. These data suggest that the HBc VLP can be an interesting carrier molecule in oral vaccine development.