One-Pot Synthesis, Purification, and Formulation of Bionanoparticle−CpG Oligodeoxynucleotide Hepatitis B Surface Antigen Conjugate Vaccine via Tangential Flow Filtration

Abstract

The synthesis and characterization of a Hepatitis B virus vaccine (HBsIC-ISS) candidate composed of Hepatitis B surface antigen (HBsAg) bionanoparticles conjugated to multiple copies of immunostimulatory sequence oligodeoxynucleotides is presented. An efficient tangential flow filtration (TFF) method has been developed to purify the conjugated bionanoparticles from the excess conjugation reagents. The TFF technique presented can serve as a rapid and convenient alternative to current methods like ultracentrifugation for the separation of excess small molecule/polymeric conjugation reagents from chemically modified viruses and other viruslike particles.

Overview and Outlook of Toll-like Receptor ligand–antigen Conjugate Vaccines

“…Several groups have evaluated the immune response to CpG-antigen conjugate vaccine candidates [102][103][104][105][106][107]. In addition to antigen delivery to immune cells by conjugation of TLR9 ligand, the presence of ODN enhances uptake of the conjugated antigen.…”

“…OVA, or CTL epitopes derived from OVA, covalently linked to CpG motifs have been shown to translocate from the endosomal route into the cytosol of DCs by receptormediated endocytosis (TLR-independent uptake) [103], inducing maturation of DCs and increasing CD40, CD86, MHC-I and MHC-II expression [106]. The antigens were then loaded onto MHC-I molecules and cross-presented to CD8 + T cells; the CpG-antigen conjugates induced CD4 + Th1 responses and protective CD8 + CTL responses, but reduced Th2 responses (allergic reactions) [102][103][104][105][106][107]. Raz and colleagues evaluated the efficacy of a CpG-conjugated HIV vaccine candidate that future science group used the protein gp120 as an antigen.…”

A promising tool for biomolecular nanotechnology - bionanoparticles

“…At the core of the particles is a bed of lipids, while the outer surface is covered in proteins. It is these proteins that can be modified for the development of custom-made vaccines (Raja et al, 2007). For example, if interested in producing a vaccine against cancer; the bacteria can be manipulated to produce bionanoparticles coated with the relevant tumour proteins (Bai and Hermans, 2007).…”

“…It is a pressure driven separation technique that utilises size selective ultrafiltration (UF) membranes or hollow fibres to separate small molecules/polymers in solution based on the difference in sizes. A novel TFF-based technique has been developed which will afford the rapid purification of poly 4-vinyl pyrrolidone (P4VP) and methyl-orange from bionanoparticles such as Qbeta (Raja et al, 2007). …”

“…The characteristic FT-IR absorption spectra (Raja et al, 2007) of lipids have been obtained using a Bruker Tensor 27 FT-IR spectrometer. All spectra are collected at 4 cm −1 spectral resolution with a scanning velocity of 20 kHz in the focused IR beam of the FT-IR interferometer.…”