Research Article

Comprehensive serological profiling of human populations using a synthetic human virome

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Science  05 Jun 2015:
Vol. 348, Issue 6239, aaa0698
DOI: 10.1126/science.aaa0698
  • Systematic viral epitope scanning (VirScan).

    This method allows comprehensive analysis of antiviral antibodies in human sera. VirScan combines DNA microarray synthesis and bacteriophage display to create a uniform, synthetic representation of peptide epitopes comprising the human virome. Immunoprecipitation and high-throughput DNA sequencing reveal the peptides recognized by antibodies in the sample. The color of each cell in the heatmap depicts the relative number of antigenic epitopes detected for a virus (rows) in each sample (columns).

  • 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) The 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) Last, amplification of the bound DNA and high-throughput sequencing of the insert DNA from bound phage reveals peptides targeted by sample antibodies. 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 (HAART) at the time the sample was taken, whereas blue dots indicate different donors before undergoing therapy. IU, international units. (D) Overlap between enriched peptides detected by VirScan and human B cell epitopes from viruses in IEDB. The entire pink circle represents the 1392 groups of nonredundant IEDB epitopes that are also present in the VirScan library (out of 1559 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 nonredundant 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 about 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 10 years of age versus over 10 years of age, (B) HIV-positive versus HIV-negative residing in the United States, (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 antivirome response recognizes a similar spectrum of peptides among infected individuals.

    In the heat-map 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 panels, 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 RSV attachment glycoprotein G (G), (B) human adenovirus C penton protein (L2), and (C) EBV 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 toward 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. Single-letter amino acid abbreviations are as follows: F, Phe; H, His; I, Ile; K, Lys; N, Asn; P, Pro; Q, Gln; R, Arg; T, Thr; V, Val; and Y, Tyr.

  • Table 1 VirScan’s sensitivity and specificity on samples with known viral infections.

    Sensitivity is the percentage of samples positive for the virus as determined by VirScan out of all n known positives. Specificity is the percentage of samples negative for the virus by VirScan out of all n known negatives.

    VirusSensitivity (n)Specificity (n)
    HCV92% (26)*97%** (34)
    HIV195% (61)*100% (33)
    HSV197% (38)100% (6)
    HSV290% (20)100% (24)

    *We found that, although the false negative samples did not meet our stringent cutoff for enriching multiple unique peptides, they had detectable antibodies to a recurrent epitope. By modifying the criterion to allow for samples that enrich multiple homologous peptides that share a recurrent epitope as described in the text, the sensitivity of detecting HCV increases to 100%, and the sensitivity for detecting HIV increases to 97%. This modified criterion does not significantly affect specificity (fig. S13). **The one false positive was from an individual whose HCV-negative status was self-reported, but who had antibodies to as many HCV peptides as 23% of the true HCV-positive individuals and is likely to be HCV-positive now or in the past. It is possible that this individual was exposed to HCV but cleared the infection. If true, the observed specificity for HCV is 100%.

    • Table 2 Frequently detected viruses.

      The % column indicates the percentage of samples that were positive for the virus by VirScan. Known HIV- and HCV-positive samples were excluded when performing this analysis.

      Virus species%
      Human herpesvirus 487.1%
      Rhinovirus B71.8%
      Human adenovirus C71.8%
      Rhinovirus A67.3%
      Human respiratory syncytial virus65.7%
      Human herpesvirus 154.4%
      Influenza A virus53.4%
      Human herpesvirus 6B52.8%
      Human herpesvirus 548.5%
      Influenza B virus40.5%
      Poliovirus33.7%
      Human herpesvirus 324.3%
      Human adenovirus F20.4%
      Human adenovirus B16.8%
      Human herpesvirus 215.5%
      Enterovirus A15.2%
      Enterovirus B13.3%

    Supplementary Materials

    • Comprehensive serological profiling of human populations using a synthetic human virome

      George J. Xu, Tomasz Kula, Qikai Xu, Mamie Z. Li, Suzanne D. Vernon, Thumbi Ndung’u, Kiat Ruxrungtham, Jorge Sanchez, Christian Brander, Raymond T. Chung, Kevin C. O’Connor, Bruce Walker, H. Benjamin Larman, Stephen J. Elledge

      Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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      • Supplementary Text
      • Figs. S1 to S14
      • Tables S1 to S3

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