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Hepatitis B virus antigen formulation for cell stimulation followed by therapeutic immunization

A technology of hepatitis B virus and antigen, applied in the field of medicine

Inactive Publication Date: 2012-09-12
CENT DE ING GENETICA & BIOTECNOLOGIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Hepatitis B virus antigen formulation for cell stimulation followed by therapeutic immunization
  • Hepatitis B virus antigen formulation for cell stimulation followed by therapeutic immunization
  • Hepatitis B virus antigen formulation for cell stimulation followed by therapeutic immunization

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1. Generation of Acid Precipitate of HBV Surface Antigen and Nucleocapsid Antigen in Suspension

[0030] HBcAg (CIGB, Cuba) was expressed as a recombinant protein of 183 amino acids, purified from Escherichia coli. Recombinant HBsAg, subtype adw2 was produced in Pichia pastoris (CIGB, Cuba). The fermentation and purification process of HBcAg protein was performed as previously described (Aguilar JC. Immunol Cell Biology 2004; 82:539-546). This antigen purification process yields highly purified nucleocapsid antigens (>95%), as figure 1 Visible in A. The molecular size of this antigen is similar to that of HBsAg, which means that virus-like particles are also formed ( figure 1 B), and additionally contains trace amounts of nucleic acid bound to its chemical structure, which functions as a TLR stimulator, such as figure 1 Visible in C. These trace amounts of bound TLR ligands can be characterized primarily as RNA and residual amounts of DNA, both of bacterial...

Embodiment 2

[0035] Example 2. Characterization of the Molecular Size of HBsAg and HBcAg Antigen Suspensions and Method of Selection of Optimal Size for Assimilation and Antigen Stimulation

[0036] In order to select the optimal size pellet for stimulating professional antigen presenting cells, 4 pellet variants were selected. Precipitate variants were produced by the method described in Example 1, dialysis and diafiltration, pH adjusted to 4.0, and incubation times predetermined.

[0037] Size selection was performed by flow cytometry, and for calibration, size-spec beads of 2.5 μm were used as an upper limit. 50 nm to 2.5 μm were used in the experiments, which demonstrated that, in general, assimilation and cell stimulation differed in dependence of pellet size and cell type. Macrophages appear to be the more readily assimilable cells, with pellet sizes greater than 500nm. In this way, the precipitate mixture was selected to increase the stimulating capacity of the antigenic mixture. ...

Embodiment 3

[0040] Example 3. Study of Passive Immunization Using Adoptive Transfer of Cells from Balb / c Mice Previously Immunized with a Preparation Containing HBsAg and HBcAg Antigens to Transgenic Balb / c Mice Expressing HBsAg

[0041] Immunotherapy based on the development of vaccine candidates constitutes one of the strategies for the treatment of chronic hepatitis B. The effectiveness of candidates depends on the generation of robust humoral and cellular immune responses capable of overcoming the state of tolerance to viral antigens that characterizes the disease.

[0042] In the present invention, anti-HBsAg and anti-HBcAg immune responses in the donor are boosted by active immunization with a formulation comprising HBsAg and HBcAg. Additionally, the cells to be transferred are activated in vitro prior to transfer so that the response to these antigens is optimal when the cells are inoculated into the recipient organism. In this artificial way, a response type that does not exist i...

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Abstract

The present invention relates to the field of therapeutic immunization, specifically with the use of a novel hepatitis B virus (HBV) antigen formulation for cell stimulation. The formulation is formed by HBV surface antigen (HBsAg) and nucleocapsid antigen (HBcAg) precipitates in suspension. The formulation contains said antigens as precipitates in suspension as particles with sizes less than 500 nm and greater than 500 nm, in a mixture in which the proportion between the particles of the sizes mentioned is between 50% - 50% and 80% - 20%, respectively. The selection of a range of particle sizes allows the levels of stimulation of various cell types to be maximized. In addition, a description is given of a method for cell stimulation using said formulation and the subsequent passive immunization of patients with chronic hepatitis B, based on the maximum in vivo or in vitro stimulation of heterologous or autologous cells (dendritic cells, B cells and macrophages). The cells stimulated with this formulation are transferred to patients with a chronic HBV infection.

Description

technical field [0001] The present invention relates to medicine, particularly therapeutic immunization by transferring cells that have been stimulated with preparations of hepatitis B virus (HBV) surface antigen (HBsAg) and nucleocapsid antigen (HBcAg). Cell stimulation was induced by formulations containing a mixture of these antigens, precipitated from suspension, in ratios selected on the basis of maximizing cell stimulation prior to passive immunotherapy application. Background technique [0002] The World Health Organization (WHO) believes that more than one-third of the world's population has been infected with hepatitis B virus (HBV). It is thought that 5% to 10% of adults and up to 90% of infected neonates develop the infection in a persistent manner, with a world level of 350,000,000 people carrying the virus. Continued replication of HBV over prolonged periods of time results in progressive liver disease that progresses to cirrhosis or liver cancer in approximate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/14A61K39/29A61P31/12
CPCA61K9/10A61K9/145A61K39/292A61K2039/545A61K2039/55505C12N2730/10134A61K2039/54A61K2039/543A61K39/12A61P31/12A61P31/14A61P31/20A61K39/464838A61K2239/38A61K2239/31A61K39/461A61K9/14A61K39/29A61K2039/5158
Inventor J·阿古拉鲁比多Y·洛拜纳马托D·赫南德兹尹古安佐H·特鲁吉洛佩尔兹F·D·L·M·弗雷尔阿尔梅达V·L·穆兹欧冈萨雷斯E·潘顿阿利亚斯S·冈萨雷斯布兰克R·乌比塔戈麦斯G·E·吉伦涅托L·S·赫里拉马丁内斯
Owner CENT DE ING GENETICA & BIOTECNOLOGIA
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