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Method for the preparation of dendritic cell vaccines

A technology of dendritic cells and vaccines, applied in the field of treatment of infectious diseases, can solve problems such as inconsistent results

Inactive Publication Date: 2014-12-24
LAB DEL DR ESTEVE SA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, reported results have been inconsistent, likely due to wide variations in the immunogen chosen, the method of inactivation, the culture and pulse conditions of the DC, and the administration regimen of the vaccine

Method used

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  • Method for the preparation of dendritic cell vaccines
  • Method for the preparation of dendritic cell vaccines
  • Method for the preparation of dendritic cell vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0211] Ex vivo generation of monocyte-derived dendritic cells (MDDCs)

[0212] 150 mL of fresh blood was drawn from a donor subject carrying HIV. Then by Ficoll density gradient (Accuspin , Sigma-Aldrich Corp., Saint Louis, MO, US) isolated peripheral blood mononuclear cells (PBMC) from blood. The resulting solution was centrifuged at 1200 rpm for 5 minutes.

[0213] The suspension was divided into 18 mL aliquots. Pour these aliquots into a 75cm 2 attached culture flask (Corning Inc., Corning, NY, US) and placed in a humidified 5% CO at 37°C 2 Atmosphere incubator for 2 to 3 hours. Unattached cells (lymphocytes) were separated by aspiration. The attached cells were mostly monocytes.

[0214] The attached cells (monocyte layer) were washed 4 times with 15 mL of X-VIVO10 (cGMP, Biowhittaker Inc., Walkersville MD, US) prewarmed at 37°C. Carefully agitate the solution to remove possible lymphocyte contamination due to gravity deposition without dislodging attached monocytes...

Embodiment 2

[0219] Maturation of autologous MDDCs was performed in flasks with attachment surfaces and

[0220] Pulse with inactivated HIV-1

[0221] After culturing for 5 days, 10.5 million MDDCs obtained in Example 1 were centrifuged at 2000 rpm for 5 minutes. Resuspend the pellet in 2.8 mL of basal medium. See Example 1. Add 0.2 mL of an aliquot of inactivated HIV that had been previously resuspended in X-VIVO15 medium containing >10 8 Copies of HIV-1 RNA. Place the cells in a 75 cm vertical and slightly inclined position 2 on a culture flask with an attachment surface. 1000 IU / mL IL-4 and 1000 IU / mL recombinant human (rh)GM-CSF (cGMP quality CellGenix GmbH, Freiburg, DE) were added to each flask and the cells were incubated at 37°C.

[0222] After incubation, add 22 mL of basal medium with GM-CSF and IL-4 at 1000 IU / mL and cytokines IL-6, TNF-α and IL-1-β at 1000 IU, 1000 IU and 300 IU per mL, respectively (cGMP quality, CellGenix GmbH, Freiburg, DE). Cells were incubated in t...

Embodiment 3

[0226] Maturation of autologous MDDCs in ultra-low attachment flasks and

[0227] Pulse with inactivated HIV-1

[0228] After 5 days of culture, 10.5 million MDDCs were centrifuged at 2000 rpm for 5 minutes. Resuspend the pellet in 2.8 mL of basal medium. See Example 1. Add 0.2 mL of an aliquot of inactivated HIV that had been previously resuspended in X-VIVO15 medium containing >10 8 Copies of HIV-1 RNA. Place the cells in a 75 cm vertical and slightly inclined position 2 culture flasks with ultra-low attachment surfaces ( , Cat. No. 153814, Cultek, SLU, Madrid, ES). Add 1000 IU / mL IL-4 and 1000 IU / mL recombinant human (rh) GM-CSF (cGMP quality CellGenix GmbH, Freiburg, DE) to each flask and incubate the cells at 37 °C with the flask in a slightly inclined position for 2 to 4 Hour.

[0229] After incubation, 22 mL of basal medium was added with 1000 IU / mL of GM-CSF and IL-4 and cytokines IL-6, TNF-α and IL-1- Beta maturation mixture. Cells were cultured in the medi...

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Abstract

The present invention relates to a process for obtaining an antigen- loaded dendritic cell showing higher viability and migratory capacity towards lymphatic nodes. The invention also relates to vaccines containing said dendritic cells as well as to the use thereof for the treatment of infectious diseases, especially AIDS.

Description

technical field [0001] The present invention relates to a method for obtaining antigen-loaded dendritic cells exhibiting higher viability and ability to migrate to lymph nodes. The present invention also relates to a vaccine comprising said dendritic cells and its use for treating infectious diseases (especially human immunodeficiency virus, HIV). Background technique [0002] While combination antiretroviral therapy (cART) is effective in inhibiting HIV-1 replication and allowing the rebuilding of CD4 T cell populations, it cannot eradicate HIV-1. Furthermore, cART failed to restore HIV-1-specific T cell immune responses. In fact, HIV-1 replication quickly rebounded to levels similar to or higher than before treatment. As a result, HIV subjects have to receive cART for life, a particularly unaffordable option considering compliance, risk of developing resistance to antiviral substances, price, and side effects, including severe metabolic abnormalities such as fat redistri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/21C12N5/0786A61P31/18C12N5/0784
CPCA61K39/4615A61K39/4611A61K39/4622A61K39/464838A61K39/4631A61K39/21A61K2039/5252C12N2501/02C12N2501/22C12N2501/2301C12N2501/2304C12N2501/2306C12N2501/25C12N2740/16034A61K39/12C12N5/0639A61P31/18A61K2239/38C12N7/00
Inventor 费莉佩·加西亚阿尔凯德特里萨·加拉特努里亚·克利门特维达尔克里斯蒂娜·吉尔罗达约瑟夫·马里亚·加特利阿蒂加斯
Owner LAB DEL DR ESTEVE SA
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