Packaging of Immunostimulatory Substances Into Virus-Like Particles: Method of Preparation and Use

a technology of immunostimulatory substances and viruses, applied in the field of vaccines, immunology and medicine, can solve the problems of unfavorable pharmacokinetics, insufficient administration of purified proteins alone, and insufficient elicitation of strong immune responses. , to achieve the effect of enhancing b and t cell responses

Inactive Publication Date: 2010-04-22
CYTOS BIOTECHNOLOGY AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017]This invention is based on the surprising finding that specific immunostimulatory substances such as DNA oligonucleotides packaged into VLPs renders them more immunogenic. Unexpectedly, the nucleic acids and oligonucleotides, respectively, present in VLPs can be replaced specifically by the immunostimulatory substances and DNA-oligonucleotides containing CpG motifs, respectively. Surprisingly, these packaged immunostimulatory substances, in particular immunostimulatory nucleic acids such as unmethylated CpG-containing oligonucleotides retained their immunostimulatory capacity without widespread activation of the innate immune system. The compositions comprising VLP's and the immunostimulatory substances in accordance with the present invention, and in particular the CpG-VLPs are dramatically more immunogenic than their CpG-free counterparts and induce enhanced B and T cell responses. The immune response against antigens optionally coupled, fused or attached otherwise to the VLPs is similarly enhanced as the immune response against the VLP itself. In addition, the T cell responses against both the VLPs and antigens are especially directed to the Th1 type. Antigens attached to CpG-loaded VLPs may therefore be ideal vaccines for prophylactic or therapeutic vaccination against allergies, tumors and other self-molecules and chronic viral diseases.
[0028]In a preferred aspect of the invention, the immune response is a T cell response, and the T cell response against the antigen is enhanced. In a specific embodiment, the T cell response is a cytotoxic T cell response, and the cytotoxic T cell response against the antigen is enhanced.

Problems solved by technology

It is well established that the administration of purified proteins alone is usually not sufficient to elicit a strong immune response; isolated antigen generally must be given together with helper substances called adjuvants.
Although DNA oligomers rich in CpG motifs can exhibit immunostimulatory capacity, their efficiency is often limited, since they are unstable in vitro and in vivo.
Thus, they exhibit unfavorable pharmacokinetics.
A second limitation for the use of CpG-oligonucleotides to stimulate immune responses is their lack of specificity, since all APC's and B cells in contact with CpG-oligonucleotides become stimulated.

Method used

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  • Packaging of Immunostimulatory Substances Into Virus-Like Particles: Method of Preparation and Use
  • Packaging of Immunostimulatory Substances Into Virus-Like Particles: Method of Preparation and Use
  • Packaging of Immunostimulatory Substances Into Virus-Like Particles: Method of Preparation and Use

Examples

Experimental program
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Effect test

example 1

[0333]Generation of p33-HBcAg VLPs.

[0334]The DNA sequence of HBcAg containing peptide p33 from LCMV is given in SEQ ID NO: 15. The p33-HBcAg VLPs were generated as follows: Hepatitis B clone pEco63 containing the complete viral genome of Hepatitis B virus was purchased from ATCC. The gene encoding HBcAg was introduced into the EcoRI / HindIII restriction sites of expression vector pkk223.3 (Pharmacia) under the control of a strong tac promoter. The p33 peptide (KAVYNFATM) (SEQ ID NO: 82) derived from lymphocytic choriomeningitis virus (LCMV) was fused to the C-terminus of HBcAg (1-185) via a three leucine-linker by standard PCR methods. 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 μl of lysozyme solution (20 mg / ml) was added. After sonication, 4 μl Benzonase and 10 mM MgCl2 was added and the suspension was incubation f...

example 2

[0338]Cloning, Expression and Purification of GA VLP

[0339]The cDNA of GA phage coat protein was amplified from GA phage by reverse transcription followed by a PCR amplification step, using the RevertAid First strand cDNA synthesis Kit (Fermentas). The cDNA was cut with the enzymes NcoI and HindIII, and cloned in vector pQβ185 previously cut with the same enzymes, leading to plasmid 355.24, harboring GA cDNA. The sequence of the inserted cDNA was checked by DNA sequencing.

[0340]Plasmid 355.24 was transformed in E. coli JM109. Expression was performed essentially as described for Qβ VLP. A single colony was inoculated in LB medium containing 20 mg / L Ampicillin overnight without shaking. This inoculum was transferred the next day into a larger flask containing M9 medium supplemented with 1% casaminoacids, 0.2% glucose and 20 mg / L Ampicillin, and incubated under shaking for 14-20 h.

[0341]GA VLP was isolated essentially as described for Qβ VLP. Cells were lysed, and the cleared lysate wa...

example 3

[0342]Fluorescein Labeled CpG-Containing Oligonucleotides can be Packaged into BKV VLPs.

[0343]VLPs produced in yeast contain small amounts of RNA which 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 small enough to enter the VLPs to eliminate the undesired ribonucleic acids. Recombinantly produced BKV VLPs (SEQ ID NO: 12) were concentrated to 1 mg / ml in PBS buffer pH7.2 and incubated in the absence or presence of RNase A (200 μg / ml, Roche Diagnostics Ltd, Switzerland) for 3 h at 37° C. After RNase A digestion BKV VLPs are supplemented with 75 nmol / ml 5′-fluorescein labeled phosphorothioate G8-8-FAM oligonucleotide (oligonucleotide from SEQ ID NO: 7) and incubated for 3 h at 37° C. Subsequently BKV VLPs are subjected to DNaseI digestion for 3 h at 37° C. (40 u / ml AMPD1, Sigma, Division of Fluka AG, Switzerland) or loaded without DNaseI digestion. The samples were complemented...

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Abstract

The invention relates to the finding that virus like particles (VLPs) can be loaded with immunostimulatory substances, in particular with DNA oligonucleotides containing non-methylated C and G (CpGs). Such CpG-VLPs are dramatically more immunogenic than their CpG-free counterparts and induce enhanced B and T cell responses. The immune response against antigens optionally coupled, fused or attached otherwise to the VLPs is similarly enhanced as the immune response against the VLP itself. In addition, the T cell responses against both the VLPs and antigens are especially directed to the Th1 type. Antigens attached to CpG-loaded VLPs may therefore be ideal vaccines for prophylactic or therapeutic vaccination against allergies, tumors and other self-molecules and chronic viral diseases.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 457,348, filed Mar. 26, 2003, which is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention is related to the fields of vaccinology, immunology and medicine. The invention provides compositions and methods for enhancing immunological responses against virus-like particles (VLPs) or against antigens coupled, fused or attached otherwise to VLPs by packaging immunostimulatory substances, in particular immunostimulatory nucleic acids, and even more particular oligonucleotides containing at least one non-methylated CpG sequence, into the VLPs. The invention can be used to induce strong and sustained T cell responses particularly useful for the treatment of tumors and chronic viral diseases as well as allergies and other chronic diseases.[0004]2. Related Art[0005]The essence of the immune s...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/12A61K39/00A61P37/04A61P31/12A61K39/21A61K39/39C07K14/02C07K14/025C07K14/08C07K14/47C12N15/117
CPCA61K39/0011C12N2740/16234A61K39/21A61K39/39A61K2039/5258A61K2039/55561A61K2039/6075C07K14/005C07K14/4748C07K2319/00C12N15/117C12N2310/18C12N2710/20022C12N2730/10122C12N2730/10123C12N2740/16222C12N2760/10022C12N2795/18122C12N2795/18123C12N2760/10034C12N2730/10134A61K39/12A61P31/12A61P35/00A61P37/04A61P37/08Y02A50/30C07K14/16
Inventor BACHMANN, MARTIN F.CORNELIUS, ANDREASMANOLOVA, VANIAMAURER, PATRIKMEIJERINK, EDWINPROBA, KARL G.SCHWARZ, KATRIN
Owner CYTOS BIOTECHNOLOGY AG
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