Screening of random peptide library of hemagglutinin from pandemic 2009 A(H1N1) influenza virus reveals unexpected antigenically important regions

Abstract

The antigenic structure of the membrane protein hemagglutinin (HA) from the 2009 A(H1N1) influenza virus was dissected with a high-throughput screening method using complex antisera. The approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (FACS) against antisera from mouse, goat and human, respectively. The amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify "hot spots" or antigenically important regions. Unexpectedly, different antigenic structures were seen for different antisera. Moreover, five antigenic regions were identified, of which all but one are located in the conserved HA stem region that is responsible for membrane fusion. Our findings are corroborated by several recent studies on cross-neutralizing H1 subtype antibodies that recognize the HA stem region. The antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory B cells and better induction of cross-neutralizing antibodies than the whole HA protein. The scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins.

Figure 1. Vectors constructed in the study.

Vector pCTCON-T was derived from pCTCON-2 by inserting an assistant sequence with two Xcm I sites at both ends between Nhe I and BamH I sites. Digestion of pCTCON-T yielded a T vector for TA ligation with random fragments of the H1N1 HA gene. ‘Stop’, ‘Stop*’ and ‘Stop**’ stand for three different stop codons for three possible ORFs, respectively. The random peptide library was expressed under the control of the galactose inducible GAL 1/10 promoter and displayed on the surface of the yeast cells by fusion to the yeast adhesion receptor AGA 2 protein. Vectors pCTCON-HA1 and pCTCON-HA2, created by inserting HA1 and HA2 genes between the Nhe I, BamH I sites of pCTCON-2, were used as positive controls in FCM detection.

Figure 2. Alignment of antigenic peptide profiles derived from screening against three antiserum samples.

(A) 56 peptides (with a RAYS ratio ≥2) obtained using mouse antisera (M-1 to M-56 from top to bottom). (B) 55 peptides obtained using goat antisera with the whole HA protein (G-1 to G-55 from the top to the bottom). (C) 51 peptides obtained using human plasma (H-1 to H-51 from top to bottom). The coordinates of the whole HA protein are indicated by the bars. The borders of the HA1 and HA2 regions (residue 345) are indicated by dashed lines.

Figure 3. FCM histograms of representative yeast clones.

The FCM signal for cells displaying antigenic peptides (the area filled by red color) overlaid with the FCM signal of non-expressing cells containing pCTCON-2 (the black curve) is shown. The designation of the clones follows the same order indicated in Fig. 2. The corresponding HA residues for each clone are illustrated. (A) FCM signals for clones M-5, M-7, M-33, and M-52 from screening against mouse antisera. (B) FCM signals for clones G-15, G-29, G-46 and G-55 from screening against goat antisera. (C) FCM signals for clones H-1, H-11, H-26 and H-45 from screening against human plasma.

Figure 4. Statistical analyses of antigenic peptides based on screening against three antiserum samples.

(A), (C) and (E) illustrate the residue frequency map for the mouse, goat and human antiserum samples, respectively. The x-axis shows amino acid residues of the HA protein. The y-axis shows the normalized frequency of individual amino acid that appears in the pool of antigenic peptides screened against each antiserum sample. (B), (D) and (F) illustrate the structural frequency map for the mouse, goat and human antiserum samples, respectively. Both the surface and backbone structure of the HA trimer of 2009 A(H1N1) virus (PDB ID: 3LZG) are annotated by colors from red to blue, representing a frequency spectrum from high to low, as shown in the color bar.

Figure 5. The five dominant antigenic regions displayed in stereo view.

The HA monomer surface view is shown on the left and colored to illustrate the five dominant antigenic regions. In order from most membrane proximal to distal: R1 (orange, residues 424–480 of HA) and R2 (yellow, residues 387–423) in HA2 were determined by screening against mouse and human antisera; R3 (purple, residues 22–60), R4 (green, residues 303–350), and R5 (cyan, residues 61–125) in HA1 were determined by screening against goat antisera. The HA monomer cartoon view is shown on the right and follows the same coloring scheme, with the third monomer shown in the back and colored in grey.

Figure 6. Binding affinities of five representative peptides to three antisera.

(A) Amino acid positions of the newly designed peptides P1–P5 (blue arrows), in relation to the five antigenic regions R1–R5. The coordinate of the HA protein is indicated by the grey bar, with the five antigenic regions represented in the same color as in Fig. 5. The peptides P1–P5 were expressed as C- terminal fusions to the thioredoxin (Trx) tag. Binding activities of the peptides against the mouse (B), goat (C) and human (D) antisera before and after immunization were characterized by the method of ELISA, with the Trx protein as the negative control. The x-axis shows the dilution ratios of corresponding antiserum samples. The y-axis shows the absorbance at 450 nm after development with the substrate 3,3′,5,5′-tetramethylbenzidine (TMB).