Comprehensive analysis of pathogen-specific antibody response in vivo based on an antigen library displayed on surface of yeast

Abstract

Host antibody response is a crucial defense against pathogenic infection. Here, we report a novel technique allowing quantitative measurement of polyclonal antibody response in vivo. This involves expression of a combinatorial library of target proteins from a candidate pathogen on the surface of yeast Saccharomyces cerevisiae. After mixing with serum/plasma from infected or immunized subjects, positive yeast clones were isolated via fluorescence-activated cell sorting (FACS). Using this technique, we have studied mouse immunized serum with recombinant hemagglutinin (HA) protein from a human influenza H5N1 strain (A/Anhui/1/2005) and convalescent plasma from an infected human in China. Our technique has identified novel antigenic domains targeted by serum/plasma and allowed calculation of the relative proportion of the antibody response against each domain. We believe such systematic measurement of an antibody response is unprecedented, and applying this method to different pathogens will improve understanding of protective immunity and guide development of vaccines and therapeutics.

FIGURE 1.

Schematic representation of experimental process for construction, selection, and analysis of combinatorial antigen library displayed on the surface of yeast S. cerevisiae.

FIGURE 2.

Construction and evaluation of combinatorial library of HA protein from a human influenza H5N1 strain (A/Anhui/1/2005) in China (A–C) and confirmation of robustness and specificity of the selection process using immunized mouse serum and a human mAb with known epitope specificity (D–E). A, generation of desired length of gene fragments for library construction by PCR amplification, DNase I digestion, and reassembling PCR technique. B, evaluation of FACS-sorted positive yeast clones under the confocal microscope. C, FACS analysis of positive single yeast clones after staining with immunized mouse serum. D, overlapping nucleotide sequences of the positive yeast clones selected and aligned to the original full-length HA sequence used for the construction of combinatorial yeast library. E, number of amino acid residues among the selected fragments along their corresponding positions in the HA protein. Bars in dark blue, red, and light blue represent the initial peptide sequences (indicated at upper left corner of each graph) used to immunize the mice and their positions relative to fragment sequences from the selected positive yeast clones. Six amino acid residues in the HA1 region critical for mAb (AVFleIgG03) binding are also indicated.

FIGURE 3.

Analysis of antigenic domains based on the positive yeast clones selected by the three immunizes serum samples sM, sA, and sB. A, overlapping nucleotide sequences of the positive yeast clones selected by serum samples sM, sA, and sB and aligned to the original full-length HA sequence used for the construction of combinatorial yeast library. B, number of amino acid residues among the selected fragments along their corresponding positions in the HA protein. C, analysis of antigenic domains in HA protein based on the short fragments using algorithms for sequence scanning and clustering. Antigenic domains are numbered and highlighted in various colors. D, no discrete antigenic domains could be identified within the long fragments due to the extended length and sequence similarity of the selected sequences. The red vertical line indicates the point where HA1 and HA2 separate. The percentages highlighted in yellow represent the AUC.

FIGURE 4.

Analysis of antigenic domains based on the positive yeast clones selected by the convalescent plasma samples from the H5N1 (A/Anhui/1/2005 or A/Vietnam/1203/04)-infected humans. Top panel shows the overlapping nucleotide sequences of the positive yeast clones (left panel) and the number of amino acid residues among the selected fragments along their corresponding positions in the HA protein (right panel) (A/Anhui/1/2005). Middle panel shows the antigenic domains in HA protein identified based on the short (left panel) and long (right panel) fragments (A/Anhui/1/2005). Bottom panel shows the antigenic domains identified based on the fragments selected from a phage display library of a different H5N1 strain (A/Vietnam/1203/04) (11). The red vertical line indicates the point where HA1 and HA2 region separate. The percentages highlighted in yellow represent the AUC.

FIGURE 5.

Structural (A) and sequence (B) analysis of antigenic domains based on the short fragments and compared with those identified using other systems. Panel a, antigenic domains D1–D7 are shown in colored patches matching that in Fig. 3C on a surface-exposed and a ribbon diagram of HA monomer within the HA trimer structure (PDB code 2IBX). Receptor binding domain (RBD) was formed by 130-Loop, 190-Helix, and 220-Loop, and domains recognized by the broadly neutralizing mAb F10 and CR6261 are shaded. Panel b, stretch of 6–23 residues at the peak of each antigenic domain (P1–P7) (within dark brackets in Table 1) is shown in side view. P1–P7 reflect the strongest recognition by the polyclonal antibody response within each antigenic domain. Panel c, top view of P2–P4 on the HA trimer structure (PDB code 2IBX) (upper panel) compared with antigenic domains Ca1, Ca2, Sa, and Sb identified for H1 glycoprotein (lower panel).

FIGURE 6.

Serum antibody responses in mice after immunization with yeast clones displaying either full-length or partial fragment of HA protein (A/Anhui/1/2005). Yeast clone displaying CD20 was used as a negative control. Numbers in parentheses indicate the starting and ending positions of displayed HA fragments (A/Anhui/1/2005).