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Improved diluent for cell-associated alphaherpesvirus vaccine

A herpes virus, herpes virus infection technology, applied in the field of vaccinology, can solve the problems of reducing the protective efficacy, not getting the full dose, loss of vaccine virus titer, etc., to achieve the effect of compensating for the loss

Pending Publication Date: 2020-08-14
INTERVET INT BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even with the production of MDV, HVT and cell-associated viruses based on HVT vectors, and the shipping of millions of doses of these vaccines this is very difficult as all processes must be optimal for the host cell and the vaccine virus
The main problems are: the availability of suitable host cells is limited, and the virus is relatively unstable
[0024] Improper storage or handling of cell-associated vaccines can lead to loss of vaccine virus titers, resulting in the target not receiving the full dose, reducing protective efficacy

Method used

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  • Improved diluent for cell-associated alphaherpesvirus vaccine
  • Improved diluent for cell-associated alphaherpesvirus vaccine
  • Improved diluent for cell-associated alphaherpesvirus vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0167] Example 1: Overview of Stability Assays

[0168] The basis of the diluent for use according to the invention is formed by phosphate buffer and sucrose.

[0169] Phosphate buffer was used at a concentration of 10 mM and set to a certain pH level by selecting the composition of the two phosphates. For example, for pH 7.3, the diluent contains: 0.324 mg / ml KH 2 PO4 and 1.356mg / ml Na 2 HPO 4 .2H 2 O.

[0170] Sucrose was used at a concentration of 50 mg / ml (ie, 150 mM), with some of the diluent samples tested having a higher sucrose content of 92.5 mg / ml (ie, 270 mM).

[0171] The concentration of phenolsulfonphthalein (Phenolsulfonphtalein) is 0.01 or 0.02 mg / ml.

[0172] In some diluent samples, with 1mM MgCl 2 Add the detected magnesium.

[0173] In some diluent samples, the CaCl tested was added at 0.133 mg / ml 2 .

[0174] In some diluent samples, the sodium citrate tested was added at 1 mM.

[0175] In some of the diluent samples tested, the following pepto...

Embodiment 2

[0180] Embodiment 2: the content detection of peptone in the diluent

[0181] In initial experiments, the effect of peptone content was examined. Recombinant HVP360-infected CEF samples were incubated for 4 hours at room temperature in diluent compositions containing varying amounts of NZ-amine. The diluent used in these experiments had a pH of 7.4 and contained magnesium and calcium ions. A series of samples were incubated in diluent plus 1% v / v NCS to simulate cell culture medium.

[0182] After incubation, samples were titrated (in triplicate) on CEF and the difference between the titers before incubation (t=0 hours) and after incubation (t=4 hours) was calculated as "delta". The standard deviation of titers is usually about 0.1. All titers are in 10 Log plaque forming units per ml.

[0183] Table 1: Effect of different contents of peptone on the in-use stability of recombinant HVT in CEF

[0184]

[0185] PB = phosphate buffer

[0186] From these results it is c...

Embodiment 3

[0187] Embodiment 3: the detection of a small amount of peptone

[0188] A follow-up experiment was performed to investigate the use of small amounts of peptone. This time the diluent did not contain magnesium or calcium, but different pH values ​​were tested. Titrations were performed in duplicate.

[0189] Table 2: Effect of small amounts of peptone on in-use stability of recombinant HVT in CEF

[0190]

[0191] Several conclusions can be drawn from these results:

[0192] - The recombinant HVT construct was found to have the greatest loss (highest delta value after 4 hr at 25°C) when maintained in standard commercial MDV diluent (with 14 mg / ml peptone)

[0193] - Minimal loss of complete medium was found

[0194] -Basic diluents of phosphate buffer and sucrose were effective in reducing titer loss of recombinant HVT in CEF for up to 4 hours at room temperature

[0195] - No need for magnesium and calcium

[0196] - pH 7.4 diluents are more effective than pH 7.2 d...

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Abstract

The present invention relates to the use of a diluent for the in-use stabilisation of cells infected with a cell-associated alphaherpesvirus. Contrary to the long-standing practice of incorporating aconsiderable amount of peptone into the diluent for such virus-infected cells, it was found that a reduction of the amount of protein in the diluent improved the in-use stability of alphaherpesvirus-infected cells. Whereby the best stability was even obtained using a protein-free diluent. The effect was especially pronounced for recombinant HVT viruses expressing a heterologous insert. Being protein-free is highly advantageous for the production of the diluent, in respect of costs, safety, and consistency of production.

Description

technical field [0001] The present invention relates to the field of vaccinology, more particularly the invention relates to the use of diluents for stabilizing in use cells infected by cell-associated alphaherpesviruses, the invention also relates to the use of anti-cell-associated alphaherpesviruses Said methods for in-use stabilization of vaccines, and methods for preparing said vaccines. Background technique [0002] Alphaherpesviruses are important pathogens of humans and virtually all animal species on Earth, and therefore of animal species associated with commercial agriculture. The preferred method of providing cheap and effective protection from infection and disease is through vaccination. For example, in commercial poultry farming, poultry are routinely vaccinated against Marek's disease and laryngotracheitis, both diseases of high concern to animal welfare and economics of operation in the poultry industry. related diseases. For example, in manuals such as "Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/12
CPCA61K39/00A61K39/12A61K2039/5254A61K2039/5256A61K2039/552A61K2039/70C12N2710/16343C12N2720/10034C12N2760/18134A61K2039/5156
Inventor A·德格罗夫I·维尔斯特根
Owner INTERVET INT BV
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