Method of purifying virus-like-particles
Inventors
Assignees
US Department of Homeland Security
Publication Number
US-11191824-B1
Publication Date
2021-12-07
Expiration Date
2041-05-20
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Abstract
A method of producing purified FMDV VLPs, comprising contacting cells containing FMDV VLPs with a lysis buffer and allowing the cells to lyse, the lysis buffer comprising 10-20 mM Tris-HCl, 150-200 mM NaCl, 3 mM MgCl2, and 1% Triton X-100, wherein the lysis buffer does not contain EDTA; centrifuging a solution; and removing a supernatant from the solution, the supernatant containing the purified FMDV VLPs.
Core Innovation
The invention provides a method of producing purified foot-and-mouth disease virus (FMDV) virus-like-particles (VLPs) by contacting cells containing FMDV VLPs with a lysis buffer comprising 10-20 mM Tris-HCl, 150-200 mM NaCl, 3 mM MgCl2, and 1% Triton X-100, wherein the buffer does not contain EDTA. After allowing cell lysis, the solution is centrifuged to remove cellular components, and the supernatant containing purified VLPs is collected. The method may include further purification by centrifuging the supernatant through a cesium chloride or sucrose gradient.
The background describes the problem of foot-and-mouth disease as a highly infectious disease affecting livestock worldwide, with vaccines currently based on chemically inactivated whole virus presenting limitations such as immunogenic instability, short shelf life, and high production costs. New vaccine generations use VLPs produced by expression of viral structural proteins. Efficient and cost-effective purification of these VLPs remains a challenge addressed by this invention.
The invention's method for purifying FMDV VLPs achieves increased purified VLP yields at a lower cost compared to previous methods, such as those using commercial lysis buffers containing EDTA, which underperform. The lysis buffer used avoids EDTA and includes MgCl2 and Triton X-100 to improve extraction efficiency. The purified VLPs are immunogenic and can be formulated into vaccines to induce a protective immune response in animals against FMDV.
Claims Coverage
The patent claims include three main independent claims covering the production of purified FMDV VLPs, the inoculation of animals with these VLPs, and compositions containing the purified VLPs. The inventive features focus on the composition of the lysis buffer, purification steps, and immunogenic applications.
Specific lysis buffer composition for purifying FMDV VLPs
The method of producing purified FMDV VLPs uses a lysis buffer comprising 10-20 mM Tris-HCl, 150-200 mM NaCl, 3 mM MgCl2, and 1% Triton X-100, explicitly excluding EDTA, to lyse cells containing FMDV VLPs and facilitate purification.
Purification process involving centrifugation and supernatant collection
After lysis, the solution is centrifuged, and the supernatant containing purified FMDV VLPs is removed; further purification can involve centrifugation through cesium chloride and/or sucrose gradients.
Method of inoculating animals using purified FMDV VLPs
The method includes providing purified FMDV VLPs, produced as described, and inoculating an animal with an effective amount to induce an immune response, providing protection against FMDV.
Composition comprising purified FMDV VLPs for vaccine use
A composition containing purified FMDV VLPs produced by the described method, which can be formulated as a vaccine for foot-and-mouth disease, optionally including an adjuvant.
The claims cover a novel method of purifying FMDV VLPs using a specific lysis buffer free from EDTA, methods of immunizing animals with the purified VLPs, and vaccine compositions containing these VLPs, emphasizing improved VLP yield, immunogenicity, and cost-effectiveness.
Stated Advantages
Increased amount of purified virus-like-particles obtained compared to previous methods.
Significantly lower cost of producing purified VLPs, with lysis buffer costing approximately $0.005 per dose versus about $31.00 per dose for a commonly used commercial buffer.
Purified VLPs produced by the method retain immunogenicity and effectively induce protective immune responses in animals against foot-and-mouth disease.
Documented Applications
Use of the purified FMDV VLPs to inoculate animals such as pigs, cows, goats, and sheep to induce an immune response protecting against foot-and-mouth disease virus.
Formulation of vaccines for foot-and-mouth disease containing an effective amount of purified FMDV VLPs.
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