VLP As Adjuvants

https://patents.google.com/patent/US20110070267

Generation of VLPs [0279] The DNA sequence of HBcAg containing peptide p33 from LCMV is given in SEQ ID NO: 70. The p33-HBcAg VLPs (p33-VLPs) were generated as follows: Hepatitis B clone pEco63 containing the complete viral genome of Hepatitis B virus was purchased from ATCC. The generation of the expression plasmid has been described previously (see WO 03/024481). [0280] A clone of E. coli K802 selected for good expression was transfected with the plasmid, and cells were grown and resuspended in 5 ml lysis buffer (10 mM Na2HPO4, 30 mM NaCl, 10 mM EDTA, 0.25% Tween-20, pH 7.0). 200 p.1 of lysozyme solution (20 mg/ml) was added. After sonication, 4 μl Benzonase and 10 mM MgCl2 was added and the suspension was incubation for 30 minutes at RT, centrifuged for 15 minutes at 15,000 rpm at 4° C. and the supernatant was retained. [0281] Next, 20% (w/v) (0.2 g/ml lysate) ammonium sulfate was added to the supernatant. After incubation for 30 minutes on ice and centrifugation for 15 minutes at 20,000 rpm at 4° C. the supernatant was discarded and the pellet resuspended in 2-3 ml PBS. 20 ml of the PBS-solution was loaded onto a Sephacryl S-400 gel filtration column (Amersham Pharmacia Biotechnology AG), fractions were loaded onto a SDS-Page gel and fractions with purified p33-HBcAg VLP capsids were pooled. Pooled fractions were loaded onto a Hydroxyappatite column. Flow through (which contains purified p33-HBcAg VLP capsids) was collected. Electron microscopy was performed according to standard protocols. A representative example is shown in FIG. 1 of Storni T., et al., (2002) J. Immunol.; 168(6):2880-6. [0282] It should be noted that the VLPs containing peptide p33 were used only for reasons of convenience, and that wild-type VLPs can likewise be used in the present invention. Throughout the description the terms p33-HBcAg VLP, HBcAg-p33 VLP, p33-VLPs and HBc33 are used interchangeably. In particular, the VLPs used in Examples 1-4, 9, and 10, 18 are p33-HBcAg VLPs. Example 2 CpG-Containing Oligonucleotides can be Packaged into HBcAg VLPs [0283] Recombinant VLPs generated as described in Example 1 were run on a native agarose (1%) gel electrophoresis and stained with ethidium bromide or Coomassie blue for the detection of RNA/DNA or protein (FIG. 1). Bacterial produced VLPs contain high levels of single stranded RNA, which is presumably binding to the arginine repeats appearing near the C-terminus of the HBcAg protein and being geographically located inside the VLPs as shown by X-ray crystallography. The contaminating RNA can be easily digested and so eliminated by incubating the VLPs with RNase A. The highly active RNase A enzyme has a molecular weight of about 14 kDa and is presumably small enough to enter the VLPs to eliminate the undesired ribonucleic acids. [0284] The recombinant VLPs were supplemented with CpG-rich oligonucleotides (see SEQ ID NO: 69) before digestion with RNase A. As shown in FIG. 2 the presence of CpG-oligonucleotides preserved the capsids structure as shown by similar migration compared to untreated p33-VLPs. The CpG-oligonucleotides containing VLPs were purified from unbound oligonucleotides via dialysis (4500-fold dilution in PBS for 24 hours using a 300 kDa MWCO dialysis membrane) (see FIG. 3). Example 3 CpG-Containing Oligonucleotides can be Packaged into VLPs by Removal of the RNA with RNAse and Subsequent Packaging of Oligonucleotides into VLPs [0285] The VLPs (containing bacterial single-stranded RNA and generated as described in Example 1) were first incubated with RNaseA to remove the RNA and in a second step the immunostimulating CpG-oligonucleotides (with normal phosphodiester moieties but also with phosphorothioate modifications of the phosphate backbone) was supplemented to the samples (FIG. 4). This experiment clearly shows that the CpG-oligonucleotides are is not absolutely required simultaneously during the RNA degradation reaction but can be added at a later time. 

Example 4 VLPs Containing CpG-Oligonucleotides Induce Strong IgG Responses Against Co-Administered Bee Venom [0286] The VLP generated as described in Example 1 was used for this experiment. Mice were subcutaneously primed with 5 μg of bee venom (ALK Abello) either alone or mixed with one of the following: 50 μg VLP alone, 50 μg VLP loaded and packaged, respectively, with CpG-oligonucleotides or 50 μg VLP mixed with 20 nmol CpG-oligonucleotides. Alternatively, mice were primed with 5 μg bee venom mixed with VLP alone or VLP loaded and packaged, respectively, with CpG-oligonucleotides in conjunction with aluminum hydroxide. 14 days later, mice were boosted with the same vaccine preparations and bled on day 21. Bee venom specific IgG responses in sera from day 21 were assessed by ELISA. RNase A treated VLPs derived from HBcAg carrying inside CpG-oligonucleotides (containing normal phosphodiester moieties), dialyzed from unbound CpG-oligonucleotides were effective at enhancing IgG responses against bee venom allergens (BV). As shown in FIG. 5, the presence of either free CpGs or VLPs loaded and packaged, respectively, with CpGs dramatically enhanced the IgG response against the bee venom. The VLP without CpGs did not enhance the immune response. The presence of Alum as an adjuvant further increased the IgG response. If IgG subclasses were measured (FIG. 6), it was evident that CpG-packaged VLPs shifted the response from an IgG1 dominance to a IgG2a dominance, indicating that a Th1 response was used. Interestingly, the presence of Alum enhanced the Th2-associated IgG1 isotype. Hence, addition of CpG-packaged VLPs to the bee venom in Alum resulted in high IgG titers but the response was still dominated by IgG1. Importantly, although CpGs packaged into VLPs were similarly effective as free CpGs at enhancing IgG responses against bee venom both in the presence or absence of Alum, they did not show signs of systemic immune activation (FIG. 7). Specifically, while vaccination of mice in the presence of free CpGs induced splenomegaly with spleens up to 4 fold increased total lymphocyte numbers, CpGs packaged into VLPs did not result in increased total lymphocyte numbers.