Rapid verification of virus particle production for a personalized vaccine
Methods for rapidly confirming production of infectious viral vectors, for use in clinical grade personalized neo-antigen vaccines for subjects in need thereof, are provided.
1. A method of quantifying the infectivity of adenoviral vector, the method comprising: transfecting a plasmid into adenoviral production cells, wherein the plasmid comprises (1) a polynucleotide encoding adenoviral proteins and (2) a polynucleotide insert encoding at least one tumor antigen;
propagating adenoviral vectors in the production cells;
purifying the adenoviral vectors by lysing the production cells and passing the lysate through an ion-exchange membrane to form a purified lysate;
loading the purified lysate onto an optical biosensor comprising a glass surface and an anti-hexon monoclonal antibody;
measuring biomass accumulation on the glass surface by bio-layer interferometry (BLI), wherein the accumulation correlates to a buildup of plasmid-transfected hexon positive production cells; and,
quantifying hexon positive production cells, wherein quantification of the purified lysate by BLI occurs within 5-30 minutes, and is correlated with infectivity of the adenoviral vector.
2. The method of claim 1 , wherein the adenoviral vectors are propagated in the production cells for 40-96 hours.
3. The method of claim 1 , wherein the adenoviral vectors are propagated in the production cells for 10 days.
4. The method of claim 1 , wherein the quantification determines the infectivity of the adenoviral vector.
5. The method of claim 1 , wherein the quantification yields an infectivity value in infectious units (IFU)/ml.
6. The method of claim 1 , further comprising incubating quantified adenoviral vectors with a suspension cell line.
7. The method of claim 6 , wherein the suspension cell is an E.C7 cell grown in a serum-free media.
8. The method of claim 6 , wherein the suspension cell is a bone marrow-derived cell, a T-lymphoblast-derived cell, or a T cell lymphoma.
9. The method of claim 8 , wherein the bone marrow-derived cell is a K-562 cell.
10. The method of claim 8 , wherein the T-lymphoblast cell is a MOLT-4 cell.
11. The method of claim 8 , wherein the T cell lymphoma is a Jurkat E6-1.
12. The method of claim 8 , wherein the bone marrow-derived cell, the T- lymphoblast-derived cell, or the T cell lymphoma is transfected with adenovirus 5 pol, pTP, Ela, and E1b.