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 producing a neoantigen vector for use in generating a neoantigen vaccine for a subject in need thereof, 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-derived neoantigen, thereby producing and propagating adenoviral vectors in the adenoviral production cells;
lysing the adenoviral production cells, wherein the lysate comprises the adenoviral vectors and adenovirus-associated hexon protein;
loading the 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 with adenovirus-producing hexon positive production cells;
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;
and thereby confirming production of infective adenoviral vectors for further propagation in a suspension cell line.
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 an alternative suspension cell, wherein the alternative suspension cell is transfected with adenovirus 5 pol, pTP, E1a, and E1b.
9 . The method of claim 8 , wherein the alternative suspension cell is a K-562 cell, a MOLT-4 cell, or a Jurkat E6-1 cell.
10 . The method of claim 6 , wherein hexon expression in the suspension cells is quantified by flow cytometry.
11 . The method of claim 10 , wherein a multiplicity of infection (MOI) is determined.
12 . The method of claim 10 , wherein the suspension cells are lysed and the adenoviral vectors are purified from the lysate by passing the lysate through an ion-exchange based separation mechanism; wherein a purified lysate is formed; and wherein the purified vectors are formulated for administration to a patient in need thereof.
13 . The method of claim 12 , wherein the ion-exchange based separation mechanism comprises an ion exchange membrane.