EXPRESSION AND CONFORMATIONAL ANALYSIS OF ENGINEERED INFLUENZA HEMAGGLUTININ
The present invention provides, among other things, compositions and methods for analyzing the expression and conformation of engineered influenza hemagglutinin In particular, the present invention provides methods of screening in silico designed HA antigens using neutralizing antibody panels specific to conserved epitopes. In some embodiments, the HAs are down selected for inclusion in universal influenza vaccines based upon their binding to neutralizing antibody panels in immunostaining assays.
1 . A method of analyzing expression and conformation of engineered hemagglutinin (HA) antigens, comprising steps of
(a) providing one or more cells, each cell comprising a nucleic acid sequence encoding an engineered HA antigen;
(b) immunostaining of the one or more cells with a panel of neutralizing antibodies under conditions that permit the neutralizing antibodies to bind to the engineered HA antigen displayed on surface of the one or more cells, wherein the panel of neutralizing antibodies comprise a plurality of neutralizing antibodies against HA stem and a plurality of neutralizing antibodies against HA head;
(c) detecting binding levels between individual neutralizing antibodies and the engineered HA antigen displayed on the surface of the one or more cells; and
(d) determining if the engineered HA antigen is properly expressed and/or folded based on the binding levels detected between the individual neutralizing antibodies and the engineered HA antigen.
2 . (canceled)
3 . The method of claim 1 , wherein the engineered HA antigen is designed by computational approaches.
4 . The method of claim 1 , wherein the engineered HA antigen is designed based on consensus sequences among a series of HA proteins from different influenza strains.
5 . The method of claim 1 , wherein the engineered HA antigen is designed based on the deletion or rearrangement of structural domains.
6 . The method of claim 1 , wherein the engineered HA antigen is designed based on swap of structural domains derived from multiple influenza strains.
7 . The method of claim 1 , wherein the engineered HA antigen is rationally designed based on combinations of neutralizing, hemagglutinin B-cell epitope patterns derived from multiple influenza strains.
8 . The method of claim 7 , wherein the engineered HA antigen comprises cross-reactive epitopes.
9 . The method of claim 1 , wherein the panel of neutralizing antibodies comprise at least three neutralizing antibodies against HA stem and at least three neutralizing antibodies against HA head.
10 . The method of claim 1 , wherein the plurality of neutralizing antibodies against HA stem comprise antibodies that bind specifically to one or more conserved epitopes in the stem region of HA from multiple influenza strains.
11 - 17 . (canceled)
18 . The method of claim 1 , wherein the plurality of neutralizing antibodies against HA head comprise antibodies bind specifically to epitopes within 20 amino acids of the receptor-binding site.
19 . The method of claim 18 , wherein the epitopes close to the receptor-binding site correspond to the N-terminal end of the short α-helix, site Sa, site Sb, the edge of the receptor pocket, the C-terminus of the short a-helix.
20 - 25 . (canceled)
26 . The method of claim 1 , wherein the individual neutralizing antibodies or secondary antibodies recognizing the individual neutralizing antibodies are labeled with a detectable entity.
27 - 28 . (canceled)
29 . The method of claim 1 , wherein the binding levels between individual neutralizing antibodies and the engineered HA antigen displayed on the surface of the one or more cells are detected by flow cytometry.
30 . (canceled)
31 . The method of claim 1 , wherein the method further comprises a step of down-selecting the engineered HA antigen as properly expressed if the binding levels are 50% or greater compared to a wild-type benchmark for at least three neutralizing antibodies against HA stem.
32 . The method of claim 31 , wherein the wild-type benchmark is defined by the binding levels between the individual neutralizing antibodies and a wild-type HA used for engineering the engineered HA.
33 . The method of claim 1 , wherein the method further comprises a step of down-selecting the engineered HA antigen as properly folded if the binding levels are over background for at least one neutralizing antibody against HA head and at least three neutralizing antibodies against HA stem.
34 . The method of claim 33 , wherein the engineered HA antigen is down-selected as properly folded if the binding levels are at least 3 times higher over background for at least one neutralizing antibody against HA head and at least three neutralizing antibodies against HA stem.
35 - 37 . (canceled)
38 . An engineered hemagglutinin (HA) antigen down-selected by a method of claim 31 .
39 . An influenza vaccine comprising an engineered hemagglutinin (HA) antigen down-selected by the method of claim 31 .
40 - 43 . (canceled)
44 . A kit for analyzing expression and conformation of engineered hemagglutinin (HA) antigens comprising a panel of neutralizing antibodies, wherein the panel of neutralizing antobodies comprise a plurality of neutralizing antibodies against HA stem and a plurality of neutralizing antibodies against HA head.
45 - 46 . (canceled)