Pathogen-specific deep sequence-coupled biopanning: A method for surveying human antibody responses

<div><p>Identifying the targets of antibody responses during infection is important for designing vaccines, developing diagnostic and prognostic tools, and understanding pathogenesis. We developed a novel deep sequence-coupled biopanning approach capable of identifying the protein epitopes of antibodies present in human polyclonal serum. Here, we report the adaptation of this approach for the identification of pathogen-specific epitopes recognized by antibodies elicited during acute infection. As a proof-of-principle, we applied this approach to assessing antibodies to Dengue virus (DENV). Using a panel of sera from patients with acute secondary DENV infection, we panned a DENV antigen fragment library displayed on the surface of bacteriophage MS2 virus-like particles and characterized the population of affinity-selected peptide epitopes by deep sequence analysis. Although there was considerable variation in the responses of individuals, we found several epitopes within the Envelope glycoprotein and Non-Structural Protein 1 that were commonly enriched. This report establishes a novel approach for characterizing pathogen-specific antibody responses in human sera, and has future utility in identifying novel diagnostic and vaccine targets.</p></div

Generation of MS2-VLP DENV-3 antigen fragment library.

<p>(A) The DENV-3 polyprotein was used to identify all possible 10 amino acid peptides, overlapping by 9 amino acids. (B) <i>E</i>. <i>coli</i> codon optimized coding sequences were synthesized by a massively parallel microchip-based synthesis-on-chip technique (LC Sciences), and (C) then used to generate a corresponding plasmid library in the pDSP62 vector. (D) The library was then expressed as VLPs in <i>E</i>. <i>coli</i> and used in subsequent deep sequence-coupled biopanning experiments.</p

Deep sequencing results from two rounds of deep sequence-coupled iterative biopanning with serum sample 1536.

<p>(A) Fold % enrichment of each peptide is presented for the entire DENV-3 polyprotein. (B and C) E and NS1 protein regions are shown enlarged from (A). Note the change in y-axis scale.</p

Expansion and Refinement of Deep Sequence-Coupled Biopanning Technology for Epitope-Specific Antibody Responses in Human Serum

Identifying the specific epitopes targeted by antibodies elicited in response to infectious diseases is important for developing vaccines and diagnostics. However, techniques for broadly exploring the specificity of antibodies in a rapid manner are lacking, limiting our ability to quickly respond to emerging viruses. We previously reported a technology that couples deep sequencing technology with a bacteriophage MS2 virus-like particle (VLP) peptide display platform for identifying pathogen-specific antibody responses. Here, we describe refinements that expand the number of patient samples that can be processed at one time, increasing the utility of this technology for rapidly responding to emerging infectious diseases. We used dengue virus (DENV) as a model system since much is already known about the antibody response. Sera from primary DENV-infected patients (n = 28) were used to pan an MS2 bacteriophage VLP library displaying all possible 10-amino-acid peptides from the DENV polypeptide. Selected VLPs were identified by deep sequencing and further investigated by enzyme-linked immunosorbent assay. We identified previously described immunodominant regions of envelope and nonstructural protein-1, as well as a number of other epitopes. Our refinement of the deep sequence-coupled biopanning technology expands the utility of this approach for rapidly investigating the specificity of antibody responses to infectious diseases

Commonly targeted E- and NS1 peptides.

<p>(A and D) The number of patients with a peptide enriched (10-fold or greater enrichment above starting library) is indicated at each amino acid position for E (A) and NS1 (D). Bars are indicated with letters are peptides enriched in 5 or more patients to highlight the commonly targeted epitopes (A and D). Individual E (B) and NS1 (E) peptides targeted by at least 5 patients are shown and colors correspond to the trimer (fusion) conformation of E (C) or NS1 (F). Dotted black lines in (F) represent the putative disordered region in which the LKYSWKTWGKAK epitope is located and is shown in (D) with an arrow. Colors of residues in (A) correspond to the colors of residues in the structure shown in (C). The junction of domain III and the stem region of E is indicated by an open arrow.</p

DENV-3 plasmid starting library.

<p>DENV-3 antigen fragment plasmid library was assessed by Ion Torrent Deep Sequencing to determine coverage at each amino acid position. (A) Deduced peptide sequences were aligned to the DENV-3 polyprotein with NCBI BLAST and used to determine the coverage at each amino acid position. Each peak represents the % total population of reads representing peptides that align starting at that amino acid position. The red and blue regions represents the E protein and NS1 protein, respectively. (B and C) The E protein and NS1 protein coverage is enlarged from (A) in order to show the coverage of the plasmid library for these regions.</p

NS1 and E enriched epitopes identified by deep sequence-coupled biopanning with patient serum after two rounds of biopanning.

<p>NS1 conservation (A) is shown for the highly enriched overlapping peptides from patient 1536 after two rounds of biopanning. Bold letters indicate the flanking amino acids that are solved in the crystal structure (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171511#pone.0171511.g005" target="_blank">Fig 5F</a> for structure). E conservation (C) is shown for the most enriched overlapping peptides from patient 1536 after two rounds of biopanning. ELISAs against synthetic peptides representing the NS1 epitope (B) and the E epitope (D) are shown. Open circles represent the data point for patient 1536 (the sample used to identify the epitopes). Primary serum samples (n = 31) and secondary serum samples (n = 30) isolated from patients 7 days post onset of fever were used.</p

Clinical Manifestations of Punta Toro Virus Species Complex Infections, Panama, 2009

An investigation in Panama found that Punta Toro virus species complex (PTVs) may contribute to febrile illnesses with symptoms mirroring those of dengue fever. However, further studies are needed to determine if PTV infection causes only a mild disease or if it can have more serious manifestations in some patients