Viral immunology. Comprehensive serological profiling of human populations using a synthetic human virome

Abstract

The human virome plays important roles in health and immunity. However, current methods for detecting viral infections and antiviral responses have limited throughput and coverage. Here, we present VirScan, a high-throughput method to comprehensively analyze antiviral antibodies using immunoprecipitation and massively parallel DNA sequencing of a bacteriophage library displaying proteome-wide peptides from all human viruses. We assayed over 10(8) antibody-peptide interactions in 569 humans across four continents, nearly doubling the number of previously established viral epitopes. We detected antibodies to an average of 10 viral species per person and 84 species in at least two individuals. Although rates of specific virus exposure were heterogeneous across populations, antibody responses targeted strongly conserved "public epitopes" for each virus, suggesting that they may elicit highly similar antibodies. VirScan is a powerful approach for studying interactions between the virome and the immune system.

Fig. 1

General VirScan analysis of the human virome. (A) Construction of the virome peptide library and VirScan screening procedure. (a) The virome peptide library consists of 93,904 56 amino acid peptides tiling, with 28 amino acid overlap, across the proteomes of all known human viruses. (b) 200 nt DNA sequences encoding the peptides were printed on a releasable DNA microarray. (c) The released DNA was amplified and cloned into a T7 phage display vector and packaged into virus particles displaying the encoded peptide on its surface. (d) The library is mixed with a sample containing antibodies that bind to their cognate peptide antigen on the phage surface. (e) The antibodies are immobilized and unbound phage are washed away. (f) Finally, amplification of the bound DNA and high throughput sequencing of the insert DNA from bound phage reveals peptides targeted by sample antibodies. Abbreviations: aa, amino acid; Ab, antibody; IP: immunoprecipitation. (B) Antibody profile of randomly chosen group of donors to show typical assay results. Each row is a virus, each column is a sample. The label above each chart indicates whether the donors are over 10 years of age or at most 10 years of age. The color intensity of each cell indicates the number of peptides from the virus that were significantly enriched by antibodies in the sample. (C) Scatter plot of the number of unique enriched peptides (after applying maximum parsimony filtering) detected in each sample against the viral load in that sample. Data are shown for the HCV positive and HIV positive samples for which we were able to obtain viral load data. For the HIV positive samples, red dots indicate samples from donors currently on highly active anti-retroviral therapy at the time the sample was taken, whereas blue dots indicate different donors prior to undergoing therapy. (D) Overlap between enriched peptides detected by VirScan and human B cell epitopes from viruses in IEDB. The entire pink circle represents the 1,392 groups of non-redundant IEDB epitopes that are also present in the VirScan library (out of 1,559 clusters total). The overlap region represents the number of groups with an epitope that is also contained in an enriched peptide detected by VirScan. The purple only region represents the number of non-redundant enriched peptides detected by VirScan that do not contain an IEDB epitope. Data are shown for peptides enriched in at least one (left) or at least two (right) samples. (E) Overlap between enriched peptides detected by VirScan and human B cell epitopes in IEDB from common human viruses. The regions represent the same values as in (D) except only epitopes corresponding to the indicated virus are considered, and only peptides from that virus that were enriched in at least two samples were considered. (F) Distribution of number of viruses detected in each sample. The histogram depicts the frequency of samples binned by the number of virus species detected by VirScan. The mean and median of the distribution are both approximately 10 virus species.

Fig. 2

Population stratification of the human virome immune response. The bar graphs depict the differences in exposure to viruses between donors who are (A) less than ten years of age versus over ten years of age, (B) HIV positive versus HIV negative, (C) residing in Peru versus residing in the United States, (D) residing in South Africa versus residing in the United States, and (E) residing in Thailand versus residing in the United States. Asterisks indicate false discovery rate < 0.05.

Fig. 3

The human anti-virome response recognizes a similar spectrum of peptides among infected individuals. In the heatmap charts, each row is a peptide tiling across the indicated protein and each column is a sample. The colored bar above each column, labeled at the top of the figure, indicates the country of origin for that sample. The samples shown are a subset of individuals with antibodies to at least one peptide from the protein. The color intensity of each cell corresponds to the −log₁₀(p-value) measure of significance of enrichment for a peptide in a sample (greater values indicates stronger antibody response). Data are shown for (A) Human respiratory syncytial virus Attachment Glycoprotein G (G), (B) Human adenovirus C penton protein (L2), and (C) Epstein-Barr virus nuclear antigen 1 (EBNA1). Data shown are the mean of two replicates.

Fig. 4

Recognition of common epitopes within an antigenic peptide from human adenovirus C penton protein (L2) across individuals. Each row is a sample. Each column denotes the first mutated position for the (A) single-, (B) double-, and (C) triple-alanine mutant peptide starting with the N-terminus on the left. Each double- and triple-alanine mutant contains two or three adjacent mutations, respectively, extending towards the C-terminus from the colored cell. The color intensity of each cell indicates the enrichment of the mutant peptide relative to the wild-type. For double-mutants, the last position is blank. The same is true for the last two positions for triple-mutants. Data shown are the mean of two replicates.