METHODS OF DETERMINING VACCINATION EFFICACY, DEVICES, AND COMPOSITIONS RELATED THERETO CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application number 61/489,757 filed May 25, 2011, hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEACH

This invention was made with government support under Grants

HHSN266200700006C awarded by NIAID and 1U19AI090023-01 awarded by the NIH. The government has certain rights in the invention.

BACKGROUND

Annual vaccination is one of the most effective methods to prevent influenza.

Two types of seasonal influenza vaccines are typically used: the trivalent inactivated influenza vaccine (TIV), given by intramuscular injection and the live attenuated influenza vaccine (LAIV), administered intra-nasally. These vaccines contain strains of influenza viruses that change annually based on the results of global influenza surveillance data. The efficacy of influenza vaccine, therefore, depends on the match of antigenicity between vaccine and circulating influenza strains. Whether a specific person responds long term to a vaccination is variable. Traditionally, detection of IgG-secreting cells to an antigen is used as an indication of efficacy. Wrammert et al., Nature 453, 667-671 (2008).

However, there is a modest correlation between the quantity of IgG-secreting cells found within one week of vaccination and the extent of an adaptive immune response a month after vaccination. Thus, there is a need to identify improved methods for determining the long term efficacy of a vaccine.

Systems vaccinology has emerged as an interdisciplinary field that combines systems wide measurements and network and predictive modeling applied to vaccinology. A systems biology approach has been used to identify gene signatures that correlate with, and predict the later immune responses in humans vaccinated with the live attenuated yellow fever vaccine YF-17D. See Querec et al., Nat Immunol. 2009;10:1 16-125. YF- 17D stimulates polyvalent innate responses and adaptive immune responses. Although systems biological approaches have been used to predict the immunogenicity of YF-17, a live replicating virus, the extent to which such approaches can be applied to predicting the immunogenicity of inactivated vaccines is unknown. Whether systems approaches can predict the immunogenicity of recall responses, as in the case of influenza where the immune response to vaccination is greatly enhanced by the past history of the vaccine recipient, both in terms of prior infections and vaccinations, is unclear.

SUMMARY

This disclosure relates to diagnostics and methods, kits, and devices related thereto for determining vaccination efficacy. In certain embodiments, the disclosure contemplates methods comprising evaluating gene expression in a sample obtained from a subject that received a vaccination and analyzing the sample for specific gene signature or expression profiles that indicate efficacy. In typical embodiments, the vaccine is an influenza vaccine.

In certain embodiments, the disclosure relates to methods of determining efficacy of an influenza virus vaccination comprising measuring the expression of a statistically significant number of genes disclosed herein, e.g. Table 1, in a sample from a subject that received a vaccination. Optionally two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twenty or more, thirty or more, forty or more, fifty or more, one hundred or more, two hundred or more genes are measured in the sample providing a gene profile. The profile may be compared to predetermined criteria for an indication of vaccine efficacy. In certain embodiments, the subject is administered another vaccination, which may be the same or different than the first vaccine, e.g., if a gene signature or profile or expression pattern does not indicate seroconversion. In certain embodiments, the subject is older than 60 or 65, younger than 5, 4 or 3 or 2, immunocompromised, or regularly taking immune suppressant drugs.

In certain embodiments, the genes are selected from ANPEP, APOBEC3B, MANF, ERLEC1, C3, CAMK4, CD38, CD72, CFD, CLPTM1L, CREB5, EIF2AK3, GGH, HSP90B1, HSPA6, HYOU1, IGHG1, ITGA6, ITGAX, KDELR2, LILRA1, LILRA2, LILRA3, LILRA6, LILRB1, LILRB2, LST1, MAN1A1, NLRP12, NR4A3, PDIA4, PDIA6, PYCARD, SIGLEC10, SIGLEC7, SLAMF7, SLC35B1, TLR5,

TNFAIP2, TNFRSF17, TNFSF13, TXNDC5, UBE2J1, and XBP1

In certain embodiments, the methods comprise analyzing or measuring four or more genes wherein the genes are selected from the groups a) GGH, LILRB2, TXNDC5, and ERLEC1; b) HSPA6, LILRA3, TXNDC5, and NLRP12; c) HSPA6, TNFRSF-17, LST1, and NLRP12; d) HSPA6, TNFRSF-17, LILRA3, and SLAMF7; e) HSPA6, GGH, CFD, and ERLEC1; f) HSPA6, MANF, SIGLEC7, and LILRA6; g) LILRBl, TXNDC5, CLPTMIL, and ERLEC1; h) HSPA6, TNFRSF-17, PYCARD, and SLAMF7; i) HSPA6, APOBEC3B, LST1, and HSP90B1; and j) HSPA6, GGH, LST1, and LILRAl providing a gene profile. The profile may be compared to predetermined criteria for an indication of vaccine efficacy.

In certain embodiments, the genes are three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twenty or more selected from genes disclosed herein, provide at least two, three, four, or five genes are selected from HSPA6, MANF, LST1, TXNDC5, PYCARD, SIGLEC7, GGH, SLAMF7, TNFRSF17, ERLECl, LILRA3, NLRP12, APOBEC3B, LILRAl, CLPTMIL, CFD, LILRA6, LILRB2, ANPEP, LILRBl, HSP90B1, IGHG1, PDIA6, HYOU1. In certain embodiments, two or more are selected from HSPA6, MANF, LST1, TXNDC5,

PYCARD, SIGLEC7, GGH, SLAMF7, TNFRSF 17, and ERLEC 1. In certain

embodiments, at least one gene is HSPA6, MANF, LST1, TXNDC5, PYCARD,

SIGLEC7, GGH, SLAMF7. In certain embodiments, at least one gene is selected from HSPA6, MANF, LST1, TXNDC5, and PYCARD.

In certain embodiments, the disclosure contemplates kits comprising probes that hybridize to nucleic acids associated with genes disclosed herein. Typically the probes are used to amplify the nucleic acids for detection or the probes contain markers such as fluorescent dyes to indicate the presence of the nucleic acids.

In certain embodiments, the disclosure contemplates devices such as arrays and microarrays comprising a solid surface consisting essentially of probes that hybridized to nucleic acids associated genes disclosed herein. In certain embodiments, three, four, five, six, seven, eight, nine or ten genes are provided.

In certain embodiments, the disclosure relates to devices comprising a solid surface consisting essentially of probes that hybridized to nucleic acids associated with three, four, five, six, seven, eight, nine or ten genes selected from genes disclosed herein and including at least two, three, or four genes selected from HSPA6, MANF, LST1,

TXNDC5, PYCARD, SIGLEC7, GGH, SLAMF7, TNFRSF 17, ERLECl, LILRA3, NLRP12, APOBEC3B, LILRAl, CLPTMIL, CFD, LILRA6, LILRB2, ANPEP, LILRBl, HSP90B1, IGHG1, PDIA6, HYOU1. In certain embodiments, the devices comprises a solid surface consisting essentially of probes that hybridized to nucleic acids associated genes selected from the groups: a) GGH, LILRB2, TXNDC5, and ERLEC1; b) HSPA6, LILRA3, TXNDC5, and NLRP12; c) HSPA6, TNFRSF-17, LST1, and NLRP12; d) HSPA6, TNFRSF-17, LILRA3, and SLAMF7; e) HSPA6, GGH, CFD, and ERLEC1; f) HSPA6, MANF, SIGLEC7, and LILRA6; g) LILRB1, TXNDC5, CLPTM1L, and ERLEC1; h) HSPA6, TNFRSF-17, PYCARD, and SLAMF7; i) HSPA6, APOBEC3B, LST1, and HSP90B1; and j) HSPA6, GGH, LST1, and LILRAl .

In certain embodiments, the disclose relates to methods of determining efficacy of a vaccination comprising measuring the expression of Calcium/calmodulin-dependent kinase IV (CamklV) gene in a sample from a subject that has been administered a vaccine wherein expression of CamklV is inversely correlated to efficacy.

In certain embodiments, the disclosure relates to methods disclosed herein further comprising measuring the expression of TNFRSF17 and/or CD38 gene in the sample from a subject that has been administered a vaccine wherein expression of TNFRSF17 and CD38 is positively correlated to efficacy.

In certain embodiments, the disclosure relates to method disclosed herein wherein measuring the expression of C-X-C motif chemokine 10 (CXCL10) gene in the sample from a subject that has been administered a vaccine wherein expression of CXCL10 is negatively correlated to efficacy.

In certain embodiments, the disclosure contemplates methods disclosed wherein comprising measuring one or more genes encoding immunoglobulin parts, wherein expression of a gene encoding immunoglobulin parts is positively correlated to efficacy. In certain embodiments, the disclosure relates to methods comprising measuring one or more genes encoding immunoglobulin parts comprising measuring one or more, or two or more, or three or more, or four or more genes selected from IGH@, IGHE, IGHG3, IGHG1 and IGHD.

In certain embodiments, the disclosure contemplates methods disclosed herein further comprising measuring the expression of one or more genes encoding rearranged variable-diversity-joining immunoglobulin gene segments wherein expression of rearranged variable-diversity-joining immunoglobulin gene segments are positively correlated to efficacy.

In certain embodiments, the vaccine is an influenza vaccine. In certain embodiments, the vaccine is a trivalent inactivated influenza virus or a live attenuated influenza virus.

In certain embodiments, the sample is obtained from the subject about 3 days or between 24 hours and 6 days after administration of the vaccine or about a week after vaccination.

In certain embodiments, the expression of the gene expression is measured in a peripheral blood mononuclear cell. In certain embodiments, the peripheral blood mononuclear cell is a B-cell, T-cell, or dendritic cell. In certain embodiments, the peripheral blood mononuclear cell is a CD19 ⁺ B cell, CD14 ⁺ monocyte, CD1 lc ^hl CD123 ¹⁰ myeloid dendritic cell (mDC) and CD123 ^hi CD1 lc ^lD plasmacytoid dendritic cell (pDC).

In certain embodiments, methods disclosed herein further comprise the step of recording a normal, decreased, or increased expression of the gene.

In certain embodiments, methods disclosed herein further comprise the set of reporting the recorded expression to a medical professional, medical institution, or a subject from which the sample was obtained or representative thereof.

In certain embodiments, methods disclosed herein further comprise the step of administering a second vaccine to the subject wherein the second vaccine is the same or a different vaccine.

In certain embodiments, with regard to any of the methods disclosed herein the expression of the gene is determined by measuring the hybridization of a nucleic acid associated with the gene to a probe. In certain embodiments, the nucleic acid associated with the gene is RNA, mR A, microR A, DNA, promoter, enhancer, or silencer sequence. In certain embodiments, the expression of gene is determined by measuring a relative level of a polypeptide in the sample associated with the gene.

In certain embodiments, the disclosure contemplates methods disclosed herein further comprising measuring the expression of one or more XBP-1 target genes.

In certain embodiments, the disclosure relates to methods of determining efficacy of a vaccination comprising measuring the expression of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more genes selected from KDELR2, HYOU1, SEC11C, SSR1, TXNDC5, SEC24D, SRPR, MOGS, SPCS2, SEC23B, PCIA6, RPN1, RPN2, CAVl, PDIA4, HSP90B1, MANF, IGHM, POU2AF1, IGHAl, TNFSF13, IGL@, IGKC, POU2F2, CD38, PECAM1, TNFRSF17 in a sample from a subject that has been administered a vaccine wherein expression of the genes is positively correlated to efficacy. In certain embodiments, the genes are selected from KDELR2, HYOU1, SPCS2, PDIA6, PDIA4, HSP90B1, MANF, IGHA1, CD38, IGKC, PECAM1, and TNFRSF17.

In certain embodiments, the disclosure relates to methods of determining efficacy of a vaccination comprising measuring the expression of two or more, three or more, four or more, five or more, genes selected from SMC3, GOPC, PIGA, ATF6B, BAX, and

IL6ST in a sample from a subject that has been administered a vaccine wherein expression of the genes is negatively correlated to efficacy.

In certain embodiments, the disclosure relates to methods of determining efficacy of a vaccination comprising measuring the expression of one, two or more, three or more, four or more genes selected from C ALR, ATF6, FCGR3 A, IGH@, and TNFRSF 13B in a sample from a subject that has been administered a vaccine wherein no change in expression of the genes is correlated to efficacy.

In certain embodiments, the disclosure contemplates methods of determining efficacy of a vaccination comprising measuring the expression of one, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more genes selected from IL10, PTPN6, TCF7L2, IFNyRbeta, IFNAR1, TLR5, ITIM3, CD4, MNDA, DAPK1, IRF2, PRKACA, PECAM1, IFI30, ANPEP, TNFSF13, MAP2K, NOD2, DDX58, ITGAX, MAPK1, TYMP, CASP1, and UBASH3B in a sample from a subject that has been administered a vaccine wherein expression of the genes are positively correlated to efficacy. In certain embodiments, the genes are selected from UBASH3B, ITGAX, and PECAM1.

In certain embodiments, the disclosure contemplates methods of determining efficacy of a vaccination comprising measuring the expression of one, two or more, three or more, four or more, five or more genes selected from STAT4, CD28, JAK1, PTPN2, JUND, and IL6ST in a sample from a subject that has been administered a vaccine wherein expression of the genes are negatively correlated to efficacy.

In certain embodiments, the disclosure relates to methods of determining efficacy of a vaccination comprising measuring the expression of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more genes selected CD3G, CD8B, CD3D, ITK TRA@, ITM2A, CD8, CD3, CD28, SLC11A2, CD6, BLK, SOS2, ZNF274, WTAP, CTLA4, KLF3, importin alpha, ITPKB, CAMK4, TFAM, BEX2, RHOH, TNFRSF25, BAG4, B4GALT1, PASK, CYB5R3 in a sample from a subject that has been administered a vaccine wherein expression of the genes is negatively correlated to efficacy. In certain embodiments, the disclosure contemplates methods of determining efficacy of a vaccination comprising measuring the expression of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more genes selected from Pak, VAV, DDX58, COROIA, TNFAIP2, IFNGR2, RGS14, PTAFR, DBT, NLRP12, ENTPD1, IFNAR1, ifn gamma, Tlr, TLR5, RNR31, CASP1, PECAM1, NAIP, LILRA2, PYCARD, CD38, C19ORF10, NOD2, CBARA1, CARD 8, TNFSF13, and TNFSF17 in a sample from a subject that has been administered a vaccine wherein expression of the genes is positively correlated to efficacy. In certain embodiments, the genes are TLR5, CASP1, PYCARD, NOD2 and NAIP.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1 A-E show data on the analysis of humoral immunity to influenza vaccination, (a) HAI titers in plasma on day 28 after vaccination with TIV or LAIV, relative to baseline (day 0); results are the highest HAI response among all three influenza strains in the vaccine: low responders, no increase above twofold; high responders, fourfold or more above baseline. P < 0.0001, mean HAI response, TIV versus LAIV (t- test). (b) ELISPOT assay of influenza-specific IgG-secreting plasmablasts among PBMCs from all vaccinees at 0 and 7 d after vaccination. Each symbol represents an individual donor; small horizontal lines indicate the median (numbers adjacent median values);

dotted lines are the limit of detection, (c) Flow cytometry analysis of plasmablasts in the plasmablast gate (CD3 CD20 ^lo~neg CD ¹⁹⁺ CD27 ^hi CD38 ^hi ) in blood from subjects vaccinated with TIV or LAIV. Numbers adjacent to outlined areas indicate percent cells in the plasmablast gate, (d) Frequency of plasmablasts, assessed by flow cytometry, versus the number of influenza-specific IgG-secreting plasmablasts, assessed by ELISPOT, at day 7 after vaccination with TIV (blue) or LAIV (black), r = 0.58 (Pearson); P < 0.0001 (for Pearson correlation; two-tailed test), (e) Influenza- specific IgG-secreting plasmablasts at day 7 versus the antibody response at day 28 after vaccination with TIV. r = 0.43 (Pearson); P = 0.02 (for Pearson correlation; two-tailed test). Data are from one experiment with 56 subjects assayed in duplicate (a), 61 subjects assayed in duplicate (b) or 59 subjects assayed once (c) or were generated from data in a-c (d,e).

Figures 2A, B illustrate molecular signatures induced by vaccination with LAIV. (a) Interferon-related genes upregulated (Up) or downregulated (Down) on day 3 or 7 ('X' in key) after vaccination with LAIV relative to their expression at day 0 (colors in key): solid lines indicate direct interactions; dashed lines indicate indirect interactions, (b) Quantitative RT-PCR confirmation of the induction of key interferon-related genes (OAS1, IRF7, MX2 and STAT1) in PBMCs obtained from healthy subjects and left unstimulated (Medium) or stimulated for 24 h in vitro with LAIV, TIV or YF-17D; results are normalized to the expression of GAPDH (glyceraldehyde phosphate dehydrogenase) and are presented relative to those of unstimulated PBMCs. Data are representative of one experiment (a) or three independent experiments with one subject each (b; error bars, s.d.).

Figures 3 A-E show data and illustrate molecular signatures induced by vaccination with TIV. (a) Heat map of gene signatures of cells of the immune response, identified by meta-analysis. Expression of each gene (rows) is presented as s.d. above (red) or below (blue) the average value for that gene for all samples (columns). mDC, myeloid DC; pDC, plasmacytoid DC; NK, natural killer, (b) Enrichment for genes upregulated by TIV among genes with high expression in any PBMC subset (numbers in plot indicate enrichment (fold)), (c) Enrichment for genes upregulated by TIV among genes with high expression in B cells and also in a specific B cell subset, (d) Heat map of genes upregulated after vaccination with TIV and also with high expression in B cells (PBMCs) and ASCs (B cell subsets); 'abParts' indicates probe sets mapping to antibody variable regions, and

Affymetrix probe identifiers are provided for probe sets not annotated, (e) Enrichment for genes upregulated by LAIV among genes with high expression in any PBMC subset. *P < 10-10 (two-tailed Fisher's exact test). Data are representative of 28 experiments with 281 samples.

Figure 4 shows data and illustrations of molecular signatures that correlate with titers of antibody to TIV. (a) Heat map of probe sets (rows) and subjects (columns) whose baseline-normalized expression at day 3 (top) or day 7 (bottom) correlated with baseline- normalized antibody response at day 28 after vaccination with TIV (colors in map indicate gene expression at day 3 or 7 relative to expression at day 0). Right margin, number of probe sets with negative correlation (blue) or positive correlation (red). Probe sets that correlated with the HAI response on both day 3 and day 7 were considered 'day 7'. P < 0.05 (Pearson), (b) HAI response-correlated genes associated with the unfolded protein response (purple shading) or ASC differentiation (tan shading) and/or regulated by XBP-1 (solid and dashed lines as in Fig. 2a). P < 0.05 (Pearson), (c) Enrichment for genes (among those with high expression in any PBMC subset) whose expression on day 3 or 7 after vaccination with TIV was positively or negatively correlated with HAI titers (cutoff, P < 0.05 (Pearson)). *P < 10-10 (two-tailed Fisher's exact test), (d) Heat map of probe sets with high expression in B cells and ASCs whose baseline-normalized expression correlated with the baseline-normalized HAI response. P < 0.05 (Pearson). Data are representative of one experiment with 28 subjects.

Figure 5 illustrates and shows data on signatures that can be used to predict the antibody response induced by TIV. (a) Experimental design used to identify the early gene signatures that can be used to predict antibody responses to vaccination with TIV: the 2008-2009 trial was used as a training set to identify predictive signatures with the DAMIP model; those signatures were then tested on the data from the 2007-2008 trial (the testing set). The expression of a subset of genes in the DAMIP predictive signatures of the 2007-2008 and 2008-2009 trials was then quantified by RT-PCR in a third independent trial (2009-2010 trial); the DAMIP model was again used to confirm the predictive signatures, (b) RT-PCR confirmation of the expression of a subset of genes in the predictive signatures generated by the DAMIP model. Each symbol represents a single gene at a given time point. P < 10-11, microarray versus RT-PCR (Pearson); r = 0.68; n = 2,897 XY pairs. Data are representative of one experiment with 44 genes from 28 subjects at two time points, (c) DAMIP gene signatures identified with the 2008-2009 trial as the training set and the 2007-2008 and 2009-2010 trials as the validation sets (DAMIP model 3); the accuracy represents the number of subjects correctly classified as 'low responders' or 'high responders' (Fig. la). Data are representative of three independent experiments.

Figure 6 shows data indicating CaMKIV regulates the antibody response to vaccines against influenza, (a) HAI response at day 28 versus microarray analysis of CAMK4 mRNA in PBMCs at day 3 after vaccination with TIV in the 2008-2009 trial (left; r = -0.47 (Pearson); P = 0.016 (for Pearson correlation; two-tailed test) or the 2007- 2008 trial (right; r = -0.73 (Pearson); P = 0.024 (for Pearson correlation; two-tailed test), (b) ELISPOT analysis of influenza-specific IgG-secreting plasmablasts at day 7 versus microarray analysis of CAMK4 mRNA on PBMCs at day 3 after vaccination with TIV. (c) Immunoblot analysis of the phosphorylation (p-) of mouse CaMKIV after in vitro stimulation of splenocytes for 1 or 2 h with various doses of TIV (above lanes). (d) Immunoblot analysis of the phosphorylation of CaMKIV after in vitro stimulation of human PBMCs for 0-720 min with lipopolysaccharide (LPS) or TIV. (e) Serum antigen- specific IgGl (top) and IgG2c (bottom) responses of wild-type and Camk4 ^~/_ mice at days 7, 14 and 28 after immunization with TIV (symbols represent individual mice), presented as absorption at 450 nm (A450). *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of one trial each with 26 subjects (2008-2009) or 9 subjects (2007-2008; a), one experiment with 26 subjects (b), three experiments (c,d) or at least four

independent experiments (e).

Figure 7 illustrates gene networks correlated to antibody response post-TIV vaccination, (a) Gene network identified by Ingenuity Pathway analysis enriched in genes negatively correlated to HAI antibody response. Solid and dashed lines represent, respectively direct and indirect interactions reported for the genes. The colors represent the correlation between day 3/ day 0 (light brown label) or day 11 day 0 (blue label) fold- change expression and the day 28/ day 0 HAI antibody response, (b) Gene network identified by Ingenuity Pathway analysis enriched in genes positively correlated to HAI antibody response.

Figure 8 illustrates canonical pathways correlated to antibody response post-TIV vaccination. (A) Functional classification performed using Ingenuity Pathway Analysis of genes whose baseline normalized expression at either day 3 or day 7 is positively correlated (Pearson, P-value < 0.05) to baseline-normalized antibody response at day 28 post-TIV vaccination. (B) "Production of Nitric Oxide and Reactive Oxygen Species in Macrophages" network (adapted from Ingenuity Pathway original network) is significantly enriched in genes whose expression is positively correlated to antibody response (see legend of Fig. 2 A). Genes with expression negatively correlated to antibody response were also represented in this network.

DETAILED DISCUSSION

Terms

As used herein, "subject" refers to any animal, preferably a human patient, livestock, or domestic pet.

The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA, or a polypeptide or its precursor (e.g., proinsulin). A functional polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the polypeptide are retained.

The term "gene" also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene which are transcribed into nuclear RNA (mRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences which are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3' flanking region may contain sequences which direct the termination of transcription, posttranscriptional cleavage and polyadenylation.

The term "polynucleotide" refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The polynucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. The term "oligonucleotide" generally refers to a short length of single-stranded polynucleotide chain usually less than 30 nucleotides long, although it may also be used interchangeably with the term "polynucleotide."

The term "nucleic acid" refers to a polymer of nucleotides, or a polynucleotide, as described above. The term is used to designate a single molecule, or a collection of molecules. Nucleic acids may be single stranded or double stranded, and may include coding regions and regions of various control elements.

The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence "A-G-T," is complementary to the sequence "T-C-A." Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.

The term "hybridization" refers to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T _m of the formed hybrid, and the G:C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be "self-hybridized."

"Amplification" is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper

polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of "target" specificity. Target sequences are "targets" in the sense that they are sought to be sorted out from other nucleic acid.

Amplification techniques have been designed primarily for this sorting out.

The term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.

Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method. The term "probe" refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, that is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any "reporter molecule," so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present disclosure be limited to any particular detection system or label.

The term "target," when used in reference to the polymerase chain reaction, refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction. Thus, the "target" is sought to be sorted out from other nucleic acid sequences. A

"segment" is defined as a region of nucleic acid within the target sequence.

The term "polymerase chain reaction" ("PCR") refers to the method of K. B.

Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, that describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e.,

denaturation, annealing and extension constitute one "cycle"; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified." With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme

32

conjugate detection; incorporation of P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any

oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.

The term "gene expression" refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through

"transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase), and into protein, through "translation" of mRNA. Gene expression can be regulated at many stages in the process. "Up-regulation" or "activation" refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while "down-regulation" or "repression" refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called "activators" and "repressors," respectively.

Seasonal Influenza Vaccines in Humans

Nakaya et al, Nature Immunology, 2011,12:786-795 and supplementary information are both incorporated by reference in their entirety. Studies during the annual influenza seasons in 2007, 2008 and 2009 were conducted in which healthy young adults were vaccinated with TIV. The goal was to undertake a detailed characterization of the innate and adaptive responses to vaccination with TIV to identify putative early signatures that correlated with or predicted the later immunogenicity, and obtain insight into the mechanisms underlying immunogenicity.

These studies indicate that systems biology approaches can be used to predict the immunogenicity of an inactivated vaccine such as TIV with up to 90% accuracy. The expression at day 3 of one of the genes in the predictive signature, encoding the kinase CaMKIV, was inversely correlated with plasma hemagglutination-inhibition (HAI) antibody titers at day 28. Vaccination of CaMKIV-deficient (Camk4-/-) mice with TIV induced enhanced antigen-specific antibody titers, which demonstrated an unappreciated role for CaMKIV in the regulation of antibody responses. This disclosure contemplates mechanisms by which effective vaccines stimulate protective immune responses; however it is not intended that certain embodiments be limited by any particular mechanism. There is an increasing appreciation of the roles of the innate immune system in sensing vaccines and tuning immune responses and emerging advances in systems biology.

Querec et al, Nat. Immunol., 2009, 10, 116-125 report a systems biology approach has been used to obtain a global picture of the immune responses in humans to vaccine YF-17D against yellow fever. This approach has identified unique biomarkers (molecular signatures) used to predict the magnitude of the antigen-specific CD8+ T cell and antibody responses induced by YF-17D6, 7 and has resulted in the formulation of new hypotheses about the mechanism of action of this vaccine. However, whether such an approach could have broad utility in the identification of signatures of immunogenicity of other kinds of vaccines, particularly inactivated vaccines, and whether such signatures would be informative about the underlying mechanisms of immunity remain unknown. To address these issues, a series of studies was done over three consecutive influenza seasons. The goal of these studies was to analyze in detail the innate and adaptive immune responses to vaccination with two vaccines against influenza, TIV and LAIV, to identify early molecular signatures that can be used to predict later immune responses and to obtain insight into the mechanisms that underlie immunogenicity. According to guidelines established by the US Food and Drug Administration, seroconversion can be defined as an HAI titer of 1 :40 or more and a minimum increase of fourfold in antibody titer after vaccination. However, it often takes several weeks after vaccination to achieve this titer; therefore, the ability to predict seroconversion just a few days after vaccination and identify nonresponders would be of great value from a public health perspective. Systems biology approaches were used to identify early signatures that predict HAI titers 4 weeks after vaccination. To accomplish this goal, an interdisciplinary approach was used, including gene-expression profiling by microarray, RT-PCR and computational methods, combined with cellular and molecular biological approaches, as well as experiments involving genetically deficient mice. Data herein indicates that such a systems biology approach can indeed be used not only to identify predictive signatures but also to obtain new insights about the immunological mechanisms involved.

LAIV induces lower serum antibody response in adults than does TIV. This probably reflects the lower 'take' of LAIV because of preexisting mucosal IgA that can neutralize the virus. Nevertheless, our microarray analysis identified a large number of genes with differences in expression, most related to the type I interferon response, in the PBMCs of subjects vaccinated with LAIV. Future studies should focus on analyzing changes in the transcriptome of the nasal mucosa after vaccination with LAIV and how that correlates with or can be used to predict local antibody responses.

Among the genes induced by vaccination with TIV, enrichment for genes with high expression in ASCs was found. This result may have reflected the rapid proliferation of plasmab lasts at day 7 after vaccination; however, our microarray analysis of B cells sorted from subjects vaccinated against influenza indicated that the changes in expression observed in PBMCs could also have been derived from real transcriptional changes in B cells. The transcription factor XBP-1, which is important for the differentiation of ASCs and the unfolded protein response, and its target genes were upregulated after vaccination with TIV and correlated with IgG and HAI responses.

The signatures that can be used to predict the T cell and B cell response to one vaccine can also be used to predict such responses to another vaccine. Notably, of the 133 genes present in the 271 DAMIP gene signatures that we used to predict the antibody response to vaccination with TIV, 7 were also predictors of the antibody response to vaccination with the YF-17D vaccine against yellow fever. Genes in the predictive signatures were TNFRSF17, which encodes BCMA, a receptor for the B cell growth factor BLyS (known to have a key role in B cell differentiation), and CD38, which encodes a surface protein important in lymphocyte development. BCMA belongs to a family of molecules (BAFF, APRIL, BAFF-R and TACI) that regulate the differentiation of plasma cells and antibody production23. Notably, there were strong correlations between the expression of genes encoding APRIL, BAFF-R and TACI and the magnitude of the HAI titers in response to vaccines against influenza and the magnitude of neutralizing antibody response to YF-17D (data not shown), which suggested that this network may be critically involved in regulating antibody responses to different vaccines.

Several studies have shown that serum HAI antibody concentrations correlate with protection against influenza. Seroconversion after vaccination, commonly defined as an increase of fourfold in HAI titers, represents a useful surrogate for vaccine efficacy when applied to a population. However, this parameter may not provide the optimal prediction of protection in an individual vaccinee or a group of vaccinees. In addition, protective concentrations of antibody may vary according to the prevalent virus subtype and laboratory doing the assay. Therefore, an increase of eightfold or more in HAI response was used to classify subjects with very high antibody responses. Using this cutoff in analyses, the DAMIP method was able to identify gene signatures that predict the antibody response induced by vaccination with TIV. The validity of these gene signatures in three independent trials were confirmed. To meet the definition of seroconversion in the US Food and Drug Administration Guidance for Industry document for this field (an HAI titer of 1 :40 or more and a minimum increase of fourfold in antibody titer after vaccination), the DAMIP analysis was re-ran using an increase of fourfold as a cutoff for defining high HAI responders. Again, the DAMIP method was able to identify sets of three to four discriminatory genes with an unbiased estimate of correct classification up to 90% for the three influenza trials.

A PCR-based assay was used (instead of an assay with gene-expression chips) with only a handful of genes. This demonstrated the feasibility of designing a cost-effective, PCR-based 'vaccine chip' that can be used to predict the immunogenicity of vaccines. Thus, we have shown how systems biology approaches can be applied to elucidate the molecular mechanisms of influenza vaccines. The predictive signatures of influenza vaccine-induced antibody responses may be used in vaccine development and in the monitoring of suboptimal immune responses (in the elderly, infants or

immunocompromised populations)

EXPERIMENTAL

Antibody responses induced by TIV and LAIV

The antibody responses of 56 healthy young adults vaccinated with either LAIV (n = 28) or TIV (n = 28) were evaluated during the 2008 influenza season. HAI titers were determined for each of the three influenza strains in LAIV and TIV in the plasma of vaccinees at baseline (day 0) and at 28 d after vaccination. The magnitude of antibody responses to the vaccine (HAI response) were calculated as the maximum difference between the HAI titer at day 28 and the baseline titer (day 0) for any of the three influenza strains contained in the vaccine (Fig. 1 A). The mean HAI response of subjects vaccinated with TIV was sixfold higher than that of those vaccinated with LAIV. Among the subjects vaccinated with TIV, there was considerable variation in the magnitude of the HAI response (> 100-fold; Fig. 1 A). According to the US Food and Drug Administration Guidance for Industry document for this field, seroconversion can be defined by an HAI titer of 1 :40 or more and a minimum fourfold increase in antibody titer after vaccination. Thus, the vaccinees were operationally classified as 'low HAI responders' or 'high HAI responders' based on whether or not a fourfold increase occurred after vaccination (Fig. 1 A). Most of the subjects vaccinated with TIV (22 of 28) were classified as high HAI responders; only six were classified as low HAI responders. In contrast, most subjects vaccinated with LAIV (24 of 28) were classified as low HAI responders and only four were classified as high HAI responders (Fig. 1 A).

Antibodies are produced by antibody-secreting B cells in the blood (plasmablasts) or bone marrow and secondary lymphoid organs (fully differentiated plasma cells). High frequencies of antigen-specific plasmablasts in the blood within a few days of vaccination, reaching a peak at day 7, have been documented. To determine whether the early plasmablast response to influenza vaccination correlated with the later HAI response, the frequency of influenza-specific plasmablasts at baseline and 7 d was assessed after vaccination (Fig. lb,c). Rapid clonal expansion of influenza-specific plasmablasts was observed 7 d after vaccination with TIV, as measured by enzyme-linked immunospot (ELISPOT) assay (Fig. lb) and by flow cytometry (Fig. lc). The population expansion of circulating plasmablasts secreting immunoglobulin G (IgG) was also greater in subjects vaccinated with TIV than in those vaccinated with LAIV (Fig. lb,c). Similar results were obtained for IgA-secreting plasmablasts at day 7 after vaccination, and a very good correlation was evident between the frequency of plasmablasts as measured by ELISPOT and their frequency as measured by flow cytometry.

As a very low HAI response was detected after vaccination with LAIV, only subjects vaccinated with TIV were considered in further correlation analyses. There was a modest positive correlation between the number of IgG-secreting plasmablasts at day 7 and the HAI response at day 28 after vaccination (Fig. le). Because the frequency of plasmablasts returns to a barely detectable amount by day 14 after vaccination, this correlation suggested that the later antibody response was associated with early circulation of plasmablasts in the blood of vaccinees. Given the modest correlation (r = 0.43), there was clearly a need for more robust correlates of immunogenicity.

Molecular signatures of influenza vaccines

It was determined whether TIV and LAIV induced molecular signatures that were detectable in the blood. To identify such signatures of immunogenicity, the concentrations of key cytokines in the plasma of vaccinees on days 0, 3 and 7 after vaccination were measured by multiplex assay. Ten cytokines or chemokines were selected on the basis of their importance as key mediators of host immune responses (CCL5 (RANTES), interleukin la, interferon-a2 (IFN-a2), CCL3 (MIP-la), CCL11 (eotaxin), interleukin 12 subunit p70, IFN-γ, interleukin 1β, CXCL10 (IP-10) and CCL2 (MCP-1)). Among those, only the chemokine CXCL10 (IP-10) was significantly induced by TIV on day 3 relative to its expression on day 0 (P = 0.0189 (t-test). None of those cytokines were significantly induced or repressed by vaccination with LAIV. The concentration of CXCL10 (IP-10) at day 3 relative to its baseline concentration was negatively correlated to the HAI response at day 28 after vaccination, which suggested possible involvement of CXCL10 (IP-10) in the antibody response. However, the correlation coefficient was modest (r = -0.48), which again emphasized the need for more robust correlates of immunogenicity.

To determine in an unbiased way the expression changes induced by vaccination against influenza on a genome-wide scale, microarray analysis was done using peripheral blood mononuclear cells (PBMCs) collected from all 56 vaccinees on days 0, 3 and 7 after vaccination. The change in expression was calculated by subtracting the log ₂ expression value at day 0 from its corresponding value day 3 or 7, and filtering out genes if no increase or decrease greater than 25% (1.25 -fold) was observed in at least 20% of the vaccinees. After that step, three independent statistical tests was applied to the remaining genes and considered only genes identified by all three analyses as being differently expressed.

Transcriptome analysis of vaccinees showed that LAIV and TIV induced very different gene signatures. However, the expression of 1,445 probe sets was altered similarly by both vaccines. Among these common 'differentially expressed genes' (DEGs), ingenuity pathway analysis identified a network composed of several genes related to inflammatory and antimicrobial responses. This indicated that processes related to innate immunity may have influenced the immunogenicity of each vaccine. The expression of several interferon-related genes was altered after vaccination with LAIV but not after vaccination with TIV (Fig. 2A,B). Type 1 interferons are central components of the innate immune response to virus. Therefore, the higher expression of type I interferon-related genes may be attributed to the replication competence of LAIV. Our analysis identified genes encoding molecules closely associated with the interferon signaling pathways, such as STATl , STAT2, TLR7, IRF3 and IRF7 (Fig. 2A). Notably, the difference in expression for many interferon-related genes was greatest at day 3 after immunization with LAIV (Fig. 2A).

The gene signatures of the two influenza vaccines were compared with that of another live attenuated vaccine, the YF-17D vaccine against yellow fever. For consistency with that publication, the same stringency and criteria was applied to identify genes with differences in expression in subjects vaccinated with YF-17D, as follows: genes were filtered out if we found no increase or decrease in expression (on day 3 or 7 relative to baseline) greater than 1.41 -fold in at least 60% of the vaccinees; one-way analysis of variance was used with the Benjamini and Hochberg false-discovery-rate method with a cutoff of 0.05; and genes had to have a difference in expression in both YF-17D trials. However, this time the analysis was done at the level of the probe set instead of defining genes based on the UniGene database (National Center for Biotechnology Information). Although subjects vaccinated with YF-17D had a gene-expression profile distinct from that of those vaccinated against influenza, many interferon-related genes were commonly induced by YF-17D and LAIV. RT-PCR analysis of RNA from PBMCs stimulated in vitro with LAIV, TIV or YF-17D confirmed that interferon-related genes were

upregulated 24 h after treatment with LAIV or YF-17D but not after stimulation with TIV (Fig. 2B). Together these data demonstrated that vaccination with TIV or LAIV induced distinct molecular signatures in the blood.

Molecular signatures of sorted cell subsets

Microarray analyses was did of the gene-expression profiles of PBMCs isolated from the blood of vaccinees at baseline and at days 3 and 7 after vaccination. One confounding variable here was that the observed transcriptional changes may have resulted from new induction of gene expression or may have simply reflected the changing cellular composition of the PBMC compartment. To overcome this issue, the approach of isolating and identifying the genomic signatures of each subset in the PBMC pool was used.

Microarray experiments were done with the following four different cell types, obtained from subjects vaccinated with LAIV (n = 6) or TIV (n = 6) and sorted by flow cytometry: CD 19+ B cells, CD 14+ monocytes, CD1 lc ^hi CD123 ^lD myeloid dendritic cells (DCs) and CD123 ^hi CD1 lc ^lD plasmacytoid DCs. Total RNA was extracted, amplified and labeled from 96 sorted cell samples at baseline and day 7 and hybridized the RNA on microarray chips. Significance analysis was done of microarrays for each subset, separately comparing the values at day 7 with the corresponding baseline values. This approach identified hundreds to thousands of probe sets with differences in expression after vaccination with TIV or LAIV, which demonstrated that vaccines against influenza produced global expression changes for each cell type. In subjects vaccinated with TIV, myeloid DCs and B cells had the most DEGs. Notably, there was an enrichment for DEGs associated with plasmablasts. However, because a substantial proportion of plasmablasts die after being frozen and thawed, the DEGs observed in the B cell compartment were probably an underestimation of the DEGs associated with plasmablasts (Fig. 3). Nevertheless, one was able to identify DEGs related to antibody-secreting cells (ASCs) and the unfolded protein response in sorted B cells after immunization with TIV. To cope with the large amount of immunoglobulin proteins that are produced, ASCs must greatly increase the function of their secretion machinery, which may lead to the accumulation of misfolded proteins in the endoplasmic reticulum. In response to such stress, the cells activate intracellular signal -transduction pathways and the unfolded protein response, which protects the cells by enhancing the capacity of the secretory apparatus and by diminishing the endoplasmic reticulum load. After vaccination with TIV, upregulation of genes encoding two transcription factors, XBP-1 and ATF6B, which are central orchestrators of the unfolded protein response, was detectable in sorted B cells but not in PBMCs.

In subjects vaccinated with LAIV, in contrast to results obtained with those vaccinated with TIV, the plasmacytoid DC subset generated the most DEGs. Of the many interferon-related genes induced by LAIV (Fig. 2A), 37 were induced in at least one subset of the sorted cells. Of those, 17 and 14 were upregulated in monocytes and plasmacytoid DCs, respectively. In addition, there were 44 interferon-related genes that were induced or repressed in at least one subset of the sorted cells but not in the PBMCs. Most were upregulated in myeloid and plasmacytoid DCs. These data suggest that antigen- presenting cells may be important in the innate response to vaccination with LAIV. The large number of interferon-related genes 'missing' from the PBMC analysis may have been due to the fact that myeloid DCs and plasmacytoid DCs together represent <1% of total PBMCs.

The observations reported here indicated the type of information that can be obtained by examination of the gene-expression profiles of sorted cell types. However, evaluating the gene-expression signatures of individual subsets of cells isolated by flow cytometry presents a considerable challenge. The practical use of such an approach is very limited, both logistically (that is, the need to use freshly isolated samples to prevent the 'preferential' loss of certain cell types, such as plasmablasts and effector T cells) and financially (that is, the need for large numbers of gene chips). Therefore, as described below, an alternative strategy was devised. Meta-analysis of cell type-specific signatures

Human PBMCs consist of many different cell types, each with a distinct transcriptome. A published study has demonstrated the use of a deconvolution method to analyze cell type-specific gene expression differences in complex tissues. See Shen-Orr et al, Nature Methods 7, 287 - 289 (2010), hereby incorporated by reference in its entirety. An independent strategy was devised to discern cell type-specific transcriptional signatures with the results of the PBMC microarray analyses. A meta-analysis was done of publicly available microarray studies in which the gene-expression profiles of isolated individual cell types of PBMCs (such as T cells, B cells, monocytes, natural killer cells and so on) or B cell subsets (such as naive, memory and germinal center B cells and ASCs from blood or tonsils) had been analyzed. To avoid issues of cross-platform normalization and probe selection, only samples that hybridized to Affymetrix Human Genome U133 Plus 2.0 Arrays or Affymetrix Human Genome U133A Arrays were used in meta-analysis. Additionally, for each study, samples were manually removed based on the severity of the disease or treatment and/or the method of cell purification. Microarray data of flow cytometry-sorted plasmacytoid and myeloid DCs obtained from PBMCs of subjects before and after vaccination with TIV or LAIV were included in our meta-analysis. The expression profile of a given cell subset was compared with the expression profile of all other subsets by t-test (P < 0.05; mean change, over twofold). A gene was designated as having high expression in a particular cell type by determining the number of times the gene was upregulated in the cell type by all possible pairwise comparisons with its expression in other cell types. The genomic signatures of cells of the immune response obtained were compared by this approach (Fig. 3 A) with the genomic signatures of subjects vaccinated against influenza.

Meta-analysis confirmed that the group of genes upregulated by TIV was enriched for genes with high expression in B cells (Fig. 3B) and, among those, genes with high expression in ASCs (Fig. 3C). A heat map of the genes upregulated was prepared in ASCs after vaccination with TIV (Fig. 3D). Among the genes upregulated were those encoding 'antibody parts' (rearranged variable-diversity-joining immunoglobulin gene segments) and several other genes encoding parts of immunoglobulins (IGH@, IGHE, IGHG3, IGHG1 and IGHD), as well as TNFRSF17 (which encodes BCMA, a member of the BAFF-BLyS family of receptors, and whose expression is a feature of the predictive signatures of neutralizing antibody responses to YF-17D6). These results confirmed the results obtained by flow cytometry and ELISPOT, with which a greater frequency of IgG+ and IgA+ ASCs was observed in the blood of vaccinees at day 7 after vaccination with TIV (Fig. 1).

In addition to the ASC signature, a signature was observed composed of several genes encoding molecules that orchestrate the unfolded protein response. The large number of XBP-1 target genes with differences in expression after vaccination was consistent with a role for XBP-1 in orchestrating the differentiation of plasma cells.

Among those, genes such as ATF6, MANF, CREB3, PDIA4, DNAJB11, HSP90B1, HERPUDl and DNAJB9 encode molecules involved in the unfolded protein response.

In contrast to results obtained for TIV, analysis of the transcriptional signature induced by LAIV by meta-analysis showed considerable enrichment for genes with high expression in T cells and monocytes (Fig. 3E). Many genes were found with high expression in natural killer cells, although these results did not reach statistical

significance. Among the interferon-related genes upregulated after vaccination with LAIV (Fig. 2A), most had high expression in monocytes and natural killer cells. That result was similar to our microarray analysis of flow cytometry-sorted cells obtained from subjects vaccinated with LAIV, in which most interferon-related genes with differences in expression in PBMCs and at least one cell subset had high expression in monocytes. These results indicate that the innate immune responses can have an important role in the mechanism of action of this live attenuated virus vaccine.

Signatures that correlate with the antibody response

Vaccination with TIV induced considerable variation in the magnitude of the HAI response (Fig. 1 A). To gain insight into the potential mechanisms underlying that variation and to identify gene signatures with which one could predict the magnitude of the HAI response, early gene signatures that correlated with the B cell responses were searched for at days 7 and 28 after vaccination with TIV. Pearson correlation analysis identified 600- 1,100 probe sets that correlated, either directly or inversely, with the magnitude of the HAI response (Fig. 4A and Table 1). Among those were several genes known to be regulated by XBP-1 and to be involved in the differentiation of plasma cells and the unfolded protein response (Fig. 4B). Table 1. Genes whose expression negatively or positively correlate to fold increase in HAI titers

KRRl, small subunit (SSU) processome

11103 KRRl component, homolog (yeast) NEG NEG

687 KLF9 Kruppel-like factor 9 NEG NEG

7403 KDM6A lysine (K)-specific demethylase 6A NEG NEG

1846 DUSP4 dual specificity phosphatase 4 NEG NEG

23774 BRD1 bromodomain containing 1 NEG NEG

G protein-coupled receptor associated

9737 GPRASP1 sorting protein 1 NEG NEG glucosamine (UDP-N-acetyl)-2-

10020 GNE epimerase/N-acetylmannosamine kinase NEG NEG signal transducer and activator of

6775 STAT4 transcription 4 NEG NEG

7695 ZNF136 zinc finger protein 136 NEG NEG

3841 KPNA5 karyopherin alpha 5 (importin alpha 6) NEG NEG solute carrier family 16, member 7

9194 SLC16A7 (monocarboxylic acid transporter 2) NEG NEG transforming growth factor, beta receptor

7048 TGFBR2 II (70/80kDa) NEG NEG

7430 EZR ezrin NEG NEG

9412 MED21 mediator complex subunit 21 NEG NEG

5805 PTS 6-pyruvoyltetrahydropterin synthase NEG NEG nuclear receptor subfamily 1, group D,

9975 NR1D2 member 2 NEG NEG

7536 SF1 splicing factor 1 NEG NEG

7750 ZMYM2 zinc finger, MYM-type 2 NEG NEG calcium/ calmodulin-dependent protein

814 CAMK4 kinase IV NEG NEG protein phosphatase, Mg2+/Mn2+

5494 PPM1A dependent, 1A NEG NEG

23015 /// GOLGA8A /// golgin A8 family, member A /// golgin A8

440270 GOLGA8B family, member B NEG NEG

51176 LEF1 lymphoid enhancer-binding factor 1 NEG NEG phosphoinositide-3 -kinase, regulatory

5295 PIK3R1 subunit 1 (alpha) NEG NEG phosphoinositide-3 -kinase, regulatory

5295 PIK3R1 subunit 1 (alpha) NEG NEG phosphoinositide-3 -kinase, regulatory

5295 PIK3R1 subunit 1 (alpha) NEG NEG

23052 ENDOD1 endonuclease domain containing 1 NEG NEG

23332 CLASP 1 cytoplasmic linker associated protein 1 NEG NEG

6935 ZEB1 zinc finger E-box binding homeobox 1 NEG NEG

6832 SUPV3L1 suppressor of varl, 3 -like 1 (S. cerevisiae) NEG NEG

51663 ZFR zinc finger RNA binding protein NEG NEG

25983 NGDN neuroguidin, EIF4E binding protein NEG NEG

3727 JUND jun D proto-oncogene NEG NEG

9774 BCLAF1 BCL2-associated transcription factor 1 NEG NEG

91746 YTHDC1 YTH domain containing 1 NEG NEG family with sequence similarity 153,

202134 /// FAM153A /// member A /// family with sequence

285596 FAM153B similarity 153, member B NEG NEG dihydrolipoamide S-succinyltransferase

(E2 component of 2-oxo-glutarate

1743 DLST complex) NEG NEG

T cell receptor associated transmembrane

50852 TRAT1 adaptor 1 NEG NEG

28984 C13orfl5 chromosome 13 open reading frame 15 NEG NEG

B-cell CLL/lymphoma 1 IB (zinc finger

64919 BCL11B protein) NEG NEG

SMEK homolog 1, suppressor of mekl

55671 SMEK1 (Dictyostelium) NEG NEG

7259 TSPYL1 TSPY-like 1 NEG NEG

54800 KLHL24 kelch-like 24 (Drosophila) NEG NEG

155370 /// SBDS /// Shwachman-Bodian-Diamond syndrome

51119 SBDSP1 /// Shwachman-Bodian-Diamond NEG NEG syndrome pseudogene 1

57189 KIAA1147 KIAA1147 NEG NEG

6167 RPL37 ribosomal protein L37 NEG NEG

27125 AFF4 AF4/FMR2 family, member 4 NEG NEG

124540 MSI2 musashi homolog 2 (Drosophila) NEG NEG

84844 PHF5A PHD finger protein 5 A NEG NEG

144438 LOCI 44438 hypothetical LOCI 44438 NEG NEG nuclear receptor subfamily 1, group D,

9975 NR1D2 member 2 NEG NEG

10944 Cl lorf58 chromosome 11 open reading frame 58 NEG NEG chromobox homolog 5 (HP1 alpha

23468 CBX5 homolog, Drosophila) NEG NEG

56987 BBX bobby sox homolog (Drosophila) NEG NEG junction mediating and regulatory protein,

133746 JMY p53 cofactor NEG NEG

9584 RBM39 RNA binding motif protein 39 NEG NEG ligand dependent nuclear receptor

84458 LCOR corepressor NEG NEG

BTB and CNC homology 1 , basic leucine

60468 BACH2 zipper transcription factor 2 NEG NEG

3899 AFF3 AF4/FMR2 family, member 3 NEG NEG growth arrest-specific 5 (non-protein

60674 GAS 5 coding) NEG NEG

3841 KPNA5 karyopherin alpha 5 (importin alpha 6) NEG NEG

266812 NAP1L5 nucleosome assembly protein 1 -like 5 NEG NEG

81579 PLA2G12A phospholipase A2, group XIIA NEG NEG

55127 HEATR1 HEAT repeat containing 1 NEG NEG

55037 PTCD3 Pentatricopeptide repeat domain 3 NEG NEG small glutamine-rich tetratricopeptide

54557 SGTB repeat (TPR)-containing, beta NEG NEG

64282 PAPD5 PAP associated domain containing 5 NEG NEG

23360 FNBP4 formin binding protein 4 NEG NEG 2778 GNAS GNAS complex locus NEG NEG

80318 GKAP1 G kinase anchoring protein 1 NEG NEG

11278 KLF12 Kruppel-like factor 12 NEG NEG

2521 FUS fused in sarcoma NEG NEG

4063 LY9 lymphocyte antigen 9 NEG NEG

4820 NKTR natural killer-tumor recognition sequence NEG NEG

Protein phosphatase, Mg2+/Mn2+

5494 PPM1A dependent, 1A NEG NEG

6428 SFRS3 Splicing factor, arginine/serine-rich 3 NEG NEG

54482 CCDC76 coiled-coil domain containing 76 NEG NEG cytotoxic T-lymphocyte-associated

1493 CTLA4 protein 4 NEG NEG

3707 ITPKB Inositol 1,4,5-trisphosphate 3-kinase B NEG NEG

80169 C17orf68 chromosome 17 open reading frame 68 NEG NEG

6095 RORA RAR-related orphan receptor A NEG NEG chromodomain helicase DNA binding

1105 CHD1 protein 1 NEG NEG

58517 RBM25 RNA binding motif protein 25 NEG NEG

26959 HBP1 HMG-box transcription factor 1 NEG NEG

BTB and CNC homology 1 , basic leucine

60468 BACH2 zipper transcription factor 2 NEG NEG

N(alpha)-acetyltransferase 25, NatB

80018 NAA25 auxiliary subunit NEG NEG

CWF19-like 2, cell cycle control (S.

143884 CWF19L2 pombe) NEG NEG

55186 SLC25A36 Solute carrier family 25, member 36 NEG NEG

339541 Clorf228 chromosome 1 open reading frame 228 NEG NEG

51444 RNF138 ring finger protein 138 NEG NEG

9643 MORF4L2 Mortality factor 4 like 2 NEG NEG

9643 MORF4L2 Mortality factor 4 like 2 NEG NEG

25962 KIAA1429 KIAA1429 NEG NEG

6789 STK4 serine/threonine kinase 4 NEG NEG 56990 CDC42SE2 CDC42 small effector 2 NEG NA

94039 ZNF101 zinc finger protein 101 NEG NA

123036 TC2N tandem C2 domains, nuclear NEG NA chromodomain helicase DNA binding

1106 CHD2 protein 2 NEG NA

56650 CLDND1 claudin domain containing 1 NEG NA

UDP-GlcNAc:betaGal beta-l,3-N-

93010 B3GNT7 acetylglucosaminyltransferase 7 NEG NA

730051 ZNF814 zinc finger protein 814 NEG NA

400579 FLJ35934 FLJ35934 protein NEG NA

54819 ZCCHC10 zinc finger, CCHC domain containing 10 NEG NA neural precursor cell expressed,

121441 NEDD1 developmentally down-regulated 1 NEG NA choline/ ethanolamine phosphotransferase

10390 CEPT1 1 NEG NA

6742 SSBP1 single-stranded DNA binding protein 1 NEG NA

2521 FUS fused in sarcoma NEG NA

728613 LOC728613 programmed cell death 6 pseudogene NEG NA

Mannosyl (alpha-l,3-)-glycoprotein beta- 1,4-N-acetylglucosaminyltransferase,

11320 MGAT4A isozyme A NEG NA

7174 TPP2 tripeptidyl peptidase II NEG NA

9782 MATR3 matrin 3 NEG NA translocase of outer mitochondrial

9804 TOMM20 membrane 20 homolog (yeast) NEG NA

894 CCND2 cyclin D2 NEG NA

9774 BCLAF1 BCL2-associated transcription factor 1 NEG NA protein phosphatase 1 , catalytic subunit,

5500 PPP1CB beta isozyme NEG NA

2273 FHL1 four and a half LIM domains 1 NEG NA

56681 SARI A SARI homolog A (S. cerevisiae) NEG NA

6421 SFPQ splicing factor proline/glutamine-rich NEG NA 5903 RANBP2 RAN binding protein 2 NEG NA

55186 SLC25A36 solute carrier family 25, member 36 NEG NA protein phosphatase 1 , regulatory

5504 PPP1R2 (inhibitor) subunit 2 NEG NA

10123 ARL4C ADP-ribosylation factor-like 4C NEG NA

8883 NAE1 NEDD8 activating enzyme El subunit 1 NEG NA

65117 RSRC2 arginine/serine-rich coiled-coil 2 NEG NA tankyrase, TRF1 -interacting ankyrin-

8658 TNKS related ADP-ribose polymerase NEG NA

55837 EAPP E2F-associated phosphoprotein NEG NA

8731 RNMT RNA (guanine-7-) methyltransferase NEG NA dual specificity phosphatase 11

8446 DUSP11 (RNA/RNP complex 1 -interacting) NEG NA

9692 KIAA0391 KIAA0391 NEG NA

2308 FOXOl forkhead box 01 NEG NA

9452 ITM2A integral membrane protein 2A NEG NA

9452 ITM2A integral membrane protein 2A NEG NA deoxynucleotidyltransferase, terminal,

30836 DNTTIP2 interacting protein 2 NEG NA

6259 RYK RYK receptor-like tyrosine kinase NEG NA

51406 NOL7 nucleolar protein 7, 27kDa NEG NA family with sequence similarity 20,

9917 FAM20B member B NEG NA

11319 ECD ecdysoneless homolog (Drosophila) NEG NA

CCAAT/enhancer binding protein

10153 CEBPZ (C/EBP), zeta NEG NA metal response element binding

22823 MTF2 transcription factor 2 NEG NA

9776 KIAA0652 KIAA0652 NEG NA

9702 CEP57 centrosomal protein 57kDa NEG NA

669 BPGM 2,3-bisphosphoglycerate mutase NEG NA

10206 TRIM 13 tripartite motif-containing 13 NEG NA 4925 NUCB2 nucleobindin 2 NEG NA

360 AQP3 aquaporin 3 (Gill blood group) NEG NA

6606 /// SMN1 /// survival of motor neuron 1 , telomeric ///

6607 SMN2 survival of motor neuron 2, centromeric NEG NA

SH3 domain containing, Ysc84-like 1 (S.

26751 SH3YL1 cerevisiae) NEG NA

10208 USPL1 ubiquitin specific peptidase like 1 NEG NA

864 RUNX3 runt-related transcription factor 3 NEG NA

79161 C7orf23 chromosome 7 open reading frame 23 NEG NA

10926 DBF4 DBF4 homolog (S. cerevisiae) NEG NA phosphoinositide-3 -kinase, catalytic, alpha

5290 PIK3CA polypeptide NEG NA

6314 ATXN7 ataxin 7 NEG NA interleukin 6 signal transducer (gpl30,

3572 IL6ST oncostatin M receptor) NEG NA myeloid/lymphoid or mixed-lineage

leukemia (trithorax homolog, Drosophila);

4300 MLLT3 translocated to, 3 NEG NA

10782 ZNF274 zinc finger protein 274 NEG NA

399 PvHOH ras homolog gene family, member H NEG NA nuclear fragile X mental retardation

26747 NUFIP1 protein interacting protein 1 NEG NA protein tyrosine phosphatase, non-receptor

5775 PTPN4 type 4 (megakaryocyte) NEG NA integrin beta 3 binding protein (beta3-

23421 ITGB3BP endonexin) NEG NA

1880 GPR183 G protein-coupled receptor 183 NEG NA

9877 ZC3H11A zinc finger CCCH-type containing 11 A NEG NA

288 ANK3 ankyrin 3, node of Ranvier (ankyrin G) NEG NA

5128 CDK17 eye lin-dependent kinase 17 NEG NA

27314 RAB30 RAB30, member RAS oncogene family NEG NA

940 CD28 CD28 molecule NEG NA CD3g molecule, gamma (CD3-TCR

917 CD3G complex) NEG NA

904 CCNT1 cyclin Tl NEG NA guanine nucleotide binding protein (G

10681 GNB5 protein), beta 5 NEG NA potassium voltage-gated channel, shaker-

3738 KCNA3 related subfamily, member 3 NEG NA solute carrier family 4, sodium

9497 SLC4A7 bicarbonate cotransporter, member 7 NEG NA

CDC 14 cell division cycle 14 homolog B

8555 CDC14B (S. cerevisiae) NEG NA

81027 TUBB1 tubulin, beta 1 NEG NA

7267 TTC3 tetratricopeptide repeat domain 3 NEG NA

23015 GOLGA8A golgin A8 family, member A NEG NA

23015 GOLGA8A golgin A8 family, member A NEG NA

6431 SFRS6 splicing factor, arginine/serine-rich 6 NEG NA alpha thalassemia/mental retardation

syndrome X- linked (RAD54 homolog, S.

546 ATRX cerevisiae) NEG NA peptidylprolyl isomerase G (cyclophilin

9360 PPIG G) NEG NA

84617 TUBB6 tubulin, beta 6 NEG NA

1069 CETN2 centrin, EF-hand protein, 2 NEG NA

3842 TNPOl transportin 1 NEG NA

9126 SMC3 structural maintenance of chromosomes 3 NEG NA iron- sulfur cluster assembly 1 homolog (S.

81689 ISCA1 cerevisiae) NEG NA iron- sulfur cluster assembly 1 homolog (S.

81689 ISCA1 cerevisiae) NEG NA

ATPase, Ca++ transporting, plasma

490 ATP2B1 membrane 1 NEG NA

54861 SNR SNF related kinase NEG NA cerebellar degeneration-related protein 2,

1039 CDR2 62kDa NEG NA

9541 CIRl corepressor interacting with RBPJ, 1 NEG NA

3298 HSF2 heat shock transcription factor 2 NEG NA

Cas-Br-M (murine) ecotropic retroviral

868 CBLB transforming sequence b NEG NA amyloid beta (A4) precursor protein-

321 APBA2 binding, family A, member 2 NEG NA

5588 PR CQ protein kinase C, theta NEG NA

9589 WTAP Wilms tumor 1 associated protein NEG NA

57396 CLK4 CDC-like kinase 4 NEG NA bolA homo log 2 (E. coli) /// PI-3-kinase-

440354 /// BOLA2 /// related kinase SMG-1 pseudogene /// PI- 552900 /// LOC440354 /// 3-kinase-related kinase SMG-1

595101 LOC595101 pseudogene NEG NA

6095 RORA RAR-related orphan receptor A NEG NA

6095 RORA RAR-related orphan receptor A NEG NA

RANBP2-like and GRIP domain

84220 RGPD5 containing 5 NEG NA

2038 EPB42 erythrocyte membrane protein band 4.2 NEG NA phosphodiesterase 4D, cAMP-specific

(phosphodiesterase E3 dunce homolog,

5144 PDE4D Drosophila) NEG NA

6789 STK4 serine/threonine kinase 4 NEG NA

4068 SH2D1A SH2 domain containing 1A NEG NA

3702 ITK IL2 -inducible T-cell kinase NEG NA synuclein, alpha (non A4 component of

6622 SNCA amyloid precursor) NEG NA

COXl 1 cytochrome c oxidase assembly

1353 COX11 homolog (yeast) NEG NA

23043 TNIK TRAF2 and NCK interacting kinase NEG NA

23215 BAT2L2 HLA-B associated transcript 2-like 2 NEG NA 5411 PNN pinin, desmosome associated protein NEG NA

25957 SFRS18 splicing factor, arginine/serine-rich 18 NEG NA

94239 H2AFV H2A histone family, member V NEG NA

GRINL1A complex locus /// glutamate

145781 /// GCOM1 /// receptor, ionotropic, N-methyl D- 81488 GRINL1A aspartate-like 1A NEG NA

23253 ANKRD12 ankyrin repeat domain 12 NEG NA

23036 ZNF292 zinc finger protein 292 NEG NA

9659 PDE4DIP phosphodiesterase 4D interacting protein NEG NA

55544 RBM38 RNA binding motif protein 38 NEG NA

9728 SECISBP2L SECIS binding protein 2-like NEG NA

9728 SECISBP2L SECIS binding protein 2-like NEG NA

MYST histone acetyltransferase

23522 MYST4 (monocytic leukemia) 4 NEG NA

23376 KIAA0776 KIAA0776 NEG NA

TCDD-inducible poly(ADP-ribose)

25976 TIPARP polymerase NEG NA

55818 KDM3A lysine (K)-specific demethylase 3A NEG NA

56987 BBX bobby sox homolog (Drosophila) NEG NA

Ral GTPase activating protein, alpha

253959 RALGAPA1 subunit 1 (catalytic) NEG NA

DnaJ (Hsp40) homolog, subfamily C,

27000 DNAJC2 member 2 NEG NA protein tyrosine phosphatase, non-receptor

5771 PTPN2 type 2 NEG NA

219333 USP12 ubiquitin specific peptidase 12 NEG NA

Heterogeneous nuclear ribonucleoprotein

D (AU-rich element RNA binding protein

3184 HNRNPD l, 37kDa) NEG NA

PAS domain containing serine/threonine

23178 PASK kinase NEG NA

915 CD3D CD3d molecule, delta (CD3-TCR NEG NA complex)

9295 SFRS11 splicing factor, arginine/serine-rich 11 NEG NA

10988 METAP2 methionyl aminopeptidase 2 NEG NA

6421 SFPQ splicing factor proline/glutamine-rich NEG NA

23215 BAT2L2 HLA-B associated transcript 2-like 2 NEG NA

57134 MAN1C1 mannosidase, alpha, class 1C, member 1 NEG NA

3081 HGD homogentisate 1 ,2-dioxygenase NEG NA

3081 HGD homogentisate 1 ,2-dioxygenase NEG NA killer cell lectin-like receptor subfamily B,

3820 KLRB1 member 1 NEG NA zinc finger and BTB domain containing

7597 ZBTB25 25 NEG NA

2273 FHL1 four and a half LIM domains 1 NEG NA

3007 HIST1H1D histone cluster 1, Hid NEG NA

375248 ANKRD36 ankyrin repeat domain 36 NEG NA

7690 ZNF131 zinc finger protein 131 NEG NA

29072 SETD2 SET domain containing 2 NEG NA

3655 ITGA6 integrin, alpha 6 NEG NA

4820 NKTR natural killer-tumor recognition sequence NEG NA

56271 BEX4 brain expressed, X-linked 4 NEG NA

6955 TRA@ T cell receptor alpha locus NEG NA mitogen-activated protein kinase kinase

4216 MAP3K4 kinase 4 NEG NA

23253 ANKRD12 Ankyrin repeat domain 12 NEG NA

PAS domain containing serine/threonine

23178 PASK kinase NEG NA

27030 MLH3 mutL homolog 3 (E. coli) NEG NA

6655 SOS2 son of sevenless homolog 2 (Drosophila) NEG NA nuclear casein kinase and cyclin-

64710 NUCKS1 dependent kinase substrate 1 NEG NA synaptotagmin binding, cytoplasmic RNA

10492 SYNCPvIP interacting protein NEG NA 51070 NOSIP nitric oxide synthase interacting protein NEG NA

7570 ZNF22 zinc finger protein 22 (KOX 15) NEG NA

51320 MEX3C mex-3 homo log C (C. elegans) NEG NA

64786 TBC1D15 TBC1 domain family, member 15 NEG NA cysteine and histidine-rich domain

26973 CHORDC1 (CHORD)-containing 1 NEG NA

11179 ZNF277 zinc finger protein 277 NEG NA

54664 TMEM106B transmembrane protein 106B NEG NA zinc finger and BTB domain containing

253461 ZBTB38 38 NEG NA

55422 ZNF331 zinc finger protein 331 NEG NA

DnaJ (Hsp40) homolog, subfamily B,

79982 DNAJB14 member 14 NEG NA

100132341 KIAA0664P3 KIAA0664 pseudogene 3 NEG NA

7559 ZNF12 zinc finger protein 12 NEG NA

100287515 LOCI 00287515 similar to zinc finger protein 26 (KOX 20)

/// 7574 /// ZNF26 /// zinc finger protein 26 NEG NA

54876 DCAF16 DDB1 and CUL4 associated factor 16 NEG NA mannosyl (alpha-l,3-)-glycoprotein beta- 1,4-N-acetylglucosaminyltransferase,

11320 MGAT4A isozyme A NEG NA

55096 FLJ10213 endogenous Borna-like N element- 1 NEG NA family with sequence similarity 60,

58516 FAM60A member A NEG NA ring finger and CCCH-type zinc finger

54542 RC3H2 domains 2 NEG NA family with sequence similarity 46,

54855 FAM46C member C NEG NA

57602 USP36 ubiquitin specific peptidase 36 NEG NA

56985 C17orf48 chromosome 17 open reading frame 48 NEG NA

79066 METHOD methyltransferase 10 domain containing NEG NA

51202 DDX47 DEAD (Asp-Glu-Ala-Asp) box NEG NA polypeptide 47

family with sequence similarity 162,

26355 FAM162A member A NEG NA cytotoxic T-lymphocyte-associated

1493 CTLA4 protein 4 NEG NA iron- sulfur cluster assembly 1 homolog (S.

81689 ISCA1 cerevisiae) NEG NA

3839 KPNA3 karyopherin alpha 3 (importin alpha 4) NEG NA

55031 USP47 ubiquitin specific peptidase 47 NEG NA large subunit GTPase 1 homolog (S.

55341 LSG1 cerevisiae) NEG NA

TAF9B RNA polymerase II, TATA box

binding protein (TBP)-associated factor,

51616 TAF9B 31kDa NEG NA phosphoinositide-3 -kinase interacting

113791 PIK3IP1 protein 1 NEG NA phosphoinositide-3 -kinase interacting

113791 PIK3IP1 protein 1 NEG NA

5128 CDK17 eye lin-dependent kinase 17 NEG NA

25926 NOL11 nucleolar protein 11 NEG NA

57187 THOC2 THO complex 2 NEG NA

8725 C19orf2 Chromosome 19 open reading frame 2 NEG NA

51187 RSL24D1 ribosomal L24 domain containing 1 NEG NA polymerase (RNA) III (DNA directed)

55718 POLR3E polypeptide E (80kD) NEG NA

50861 STMN3 stathmin-like 3 NEG NA

8436 SDPR serum deprivation response NEG NA

6322 SCML1 sex comb on midleg-like 1 (Drosophila) NEG NA

54664 TMEM106B transmembrane protein 106B NEG NA

54665 RSBN1 round spermatid basic protein 1 NEG NA

DnaJ (Hsp40) homolog, subfamily B,

79982 DNAJB14 member 14 NEG NA DEAH (Asp-Glu-Ala-His) box

170506 DHX36 polypeptide 36 NEG NA

55893 ZNF395 zinc finger protein 395 NEG NA

27086 FOXP1 forkhead box PI NEG NA

56985 C17orf48 chromosome 17 open reading frame 48 NEG NA

DEAD/H (Asp-Glu-Ala- Asp/His) box

84771 DDX11L2 polypeptide 11 like 2 NEG NA

9439 MED23 mediator complex subunit 23 NEG NA nasopharyngeal carcinoma, down-

246734 NPCDR1 regulated 1 NEG NA

84707 BEX2 brain expressed X-linked 2 NEG NA

10163 WASF2 WAS protein family, member 2 NEG NA metastasis associated lung

adenocarcinoma transcript 1 (non-protein

378938 MALAT1 coding) NEG NA

81539 SLC38A1 solute carrier family 38, member 1 NEG NA small nucleolar R A host gene 1 (non¬

23642 SNHG1 protein coding) NEG NA

50808 AK3 adenylate kinase 3 NEG NA

113246 C12orf57 chromosome 12 open reading frame 57 NEG NA

3146 HMGB1 high-mobility group box 1 NEG NA

56889 TM9SF3 transmembrane 9 superfamily member 3 NEG NA

90488 C12orf23 chromosome 12 open reading frame 23 NEG NA

54602 NDFIP2 Nedd4 family interacting protein 2 NEG NA

57291 KIAA0114 KIAA0114 NEG NA

84916 CIRH1A cirrhosis, autosomal recessive 1 A (cirhin) NEG NA

112942 CCDC104 coiled-coil domain containing 104 NEG NA

387882 C12orf75 chromosome 12 open reading frame 75 NEG NA

26224 FBXL3 F-box and leucine-rich repeat protein 3 NEG NA jumonji C domain containing histone

80853 JHDM1D demethylase 1 homo log D (S. cerevisiae) NEG NA093630 SNHG8 small nucleolar RNA host gene 8 (non- NEG NA protein coding)

64645 HI ATI hippocampus abundant transcript 1 NEG NA

132299 OCIAD2 OCIA domain containing 2 NEG NA

56204 KIAA1370 KIAA1370 NEG NA glycerol-3 -phosphate acyltransferase,

57678 GPAM mitochondrial NEG NA

114926 C8orf40 chromosome 8 open reading frame 40 NEG NA

163882 CNST consortin, connexin sorting protein NEG NA

Hypothetical gene supported by

401504 LOC401504 AK091718 NEG NA hypothetical gene supported by

401504 LOC401504 AK091718 NEG NA

51755 CDK12 eye lin-dependent kinase 12 NEG NA

51755 CDK12 eye lin-dependent kinase 12 NEG NA

114915 NCRNA00219 non-protein coding RNA 219 NEG NA

90799 CCDC45 coiled-coil domain containing 45 NEG NA

388796 LOC388796 hypothetical LOC388796 NEG NA

Chromodomain helicase DNA binding

1106 CHD2 protein 2 NEG NA

153222 C5orf41 chromosome 5 open reading frame 41 NEG NA

153222 C5orf41 chromosome 5 open reading frame 41 NEG NA

CCR4-NOT transcription complex,

246175 CNOT6L subunit 6-like NEG NA

258010 SVIP small VCP/p97-interacting protein NEG NA

RPTOR independent companion of

253260 RICTOR MTOR, complex 2 NEG NA

55269 PSPC1 paraspeckle component 1 NEG NA

6095 RORA RAR-related orphan receptor A NEG NA

152137 CCDC50 coiled-coil domain containing 50 NEG NA family with sequence similarity 76,

143684 FAM76B member B NEG NA

51631 LUC7L2 LUC7-like 2 (S. cerevisiae) NEG NA family with sequence similarity 46,

54855 FAM46C member C NEG NA

130502 TTC32 tetratricopeptide repeat domain 32 NEG NA

64282 PAPD5 PAP associated domain containing 5 NEG NA

57456 KIAA1143 KIAA1143 NEG NA oxidoreductase NAD-binding domain

92106 OXNAD1 containing 1 NEG NA

5814 PURB purine-rich element binding protein B NEG NA

90390 MED30 mediator complex subunit 30 NEG NA

Sep (O-phosphoserine) tRNA:Sec

51091 SEPSECS (selenocysteine) tRNA synthase NEG NA

55728 N4BP2 NEDD4 binding protein 2 NEG NA

53344 CHIC1 cysteine-rich hydrophobic domain 1 NEG NA small nuclear ribonucleoprotein

6638 SNRPN polypeptide N NEG NA glycoprotein, alpha-galactosyltransferase

2681 GGTA1 1 pseudogene NEG NA

91298 C12orf29 chromosome 12 open reading frame 29 NEG NA activating transcription factor 7 interacting

80063 ATF7IP2 protein 2 NEG NA

TAF9B RNA polymerase II, TATA box

binding protein (TBP)-associated factor,

51616 TAF9B 31kDa NEG NA

91746 YTHDC1 YTH domain containing 1 NEG NA echinoderm microtubule associated

27436 EML4 protein like 4 NEG NA

10600 USP16 ubiquitin specific peptidase 16 NEG NA calcium/ calmodulin-dependent protein

814 CAMK4 kinase IV NEG NA

11123 RCAN3 RCAN family member 3 NEG NA

84830 C6orfl05 chromosome 6 open reading frame 105 NEG NA

3841 KPNA5 karyopherin alpha 5 (importin alpha 6) NEG NA 5094 PCBP2 Poly(rC) binding protein 2 NEG NA

SPT2, Suppressor of Ty, domain

144108 SPTY2D1 containing 1 (S. cerevisiae) NEG NA

9589 WTAP Wilms tumor 1 associated protein NEG NA

388335 TMEM220 transmembrane protein 220 NEG NA

2130 EWSR1 Ewing sarcoma breakpoint region 1 NEG NA

258010 SVIP small VCP/p97-interacting protein NEG NA

94081 SFXN1 sideroflexin 1 NEG NA

Rap guanine nucleotide exchange factor

51735 RAPGEF6 (GEF) 6 NEG NA

258010 SVIP small VCP/p97-interacting protein NEG NA

83478 ARHGAP24 Rho GTPase activating protein 24 NEG NA

439949 LOC439949 hypothetical protein LOC439949 NEG NA

4735 SEPT2 Septin 2 NEG NA

79893 GGNBP2 gametogenetin binding protein 2 NEG NA

DIS3 mitotic control homo log (S.

22894 DIS3 cerevisiae) NEG NA

123036 TC2N tandem C2 domains, nuclear NEG NA

TAF4b RNA polymerase II, TATA box

binding protein (TBP)-associated factor,

6875 TAF4B 105kDa NEG NA

112840 WDR89 WD repeat domain 89 NEG NA family with sequence similarity 169,

26049 FAM169A member A NEG NA

168374 ZNF92 zinc finger protein 92 NEG NA

SPT2, Suppressor of Ty, domain

144108 SPTY2D1 containing 1 (S. cerevisiae) NEG NA

84441 MAML2 mastermind-like 2 (Drosophila) NEG NA family with sequence similarity 133,

257415 FAM133B member B NEG NA

Elongation protein 2 homo log (S.

55250 ELP2 cerevisiae) NEG NA establishment of cohesion 1 homo log 1 (S.

114799 ESCOl cerevisiae) NEG NA protein kinase, cAMP-dependent,

5567 PRKACB catalytic, beta NEG NA protein kinase, interferon-inducible double

stranded RNA dependent activator ///

protein kinase, interferon-inducible double

731716 /// PRKRA /// stranded RNA dependent activator

8575 PRKRAP1 pseudogene 1 NEG NA

7403 KDM6A lysine (K)-specific demethylase 6A NEG NA

STT3, subunit of the

oligosaccharyltransferase complex,

201595 STT3B homo log B (S. cerevisiae) NEG NA

Heterogeneous nuclear ribonucleoprotein

D (AU-rich element RNA binding protein

3184 HNR PD l, 37kDa) NEG NA

3716 JAK1 Janus kinase 1 NEG NA

3708 ITPR1 inositol 1,4, 5-triphosphate receptor, type 1 NEG NA calcium/ calmodulin-dependent protein

814 CAMK4 kinase IV NEG NA

4008 LM07 LIM domain 7 NEG NA spectrin repeat containing, nuclear

23224 SYNE2 envelope 2 NEG NA

54737 MPHOSPH8 M-phase phosphoprotein 8 NEG NA

80821 DDHD1 DDHD domain containing 1 NEG NA

10625 IVNS1ABP influenza virus NS 1 A binding protein NEG NA

Chromodomain helicase DNA binding

1106 CHD2 protein 2 NEG NA protein phosphatase 1 , regulatory

26051 PPP1R16B (inhibitor) subunit 16B NEG NA

57473 ZNF512B zinc finger protein 512B NEG NA

159091 FAM122C family with sequence similarity 122C NA NEG 5930 RBBP6 retinoblastoma binding protein 6 NA NEG

80213 TM2D3 TM2 domain containing 3 NA NEG

T-cell activation RhoGTPase activating

117289 TAGAP protein NA NEG

80012 PHC3 polyhomeotic homolog 3 (Drosophila) NA NEG

92797 HELB helicase (DNA) B NA NEG

200765 TIGD1 tigger transposable element derived 1 NA NEG family with sequence similarity 117,

150864 FAM117B member B NA NEG

CCR4-NOT transcription complex,

246175 CNOT6L subunit 6-like NA NEG

AT rich interactive domain 2 (ARID,

196528 ARID2 RFX-like) NA NEG

7559 ZNF12 zinc finger protein 12 NA NEG heterochromatin protein 1 , binding protein

50809 HP1BP3 3 NA NEG

113510 HELQ helicase, POLQ-like NA NEG mesoderm induction early response 1 ,

166968 MIER3 family member 3 NA NEG

1388 ATF6B activating transcription factor 6 beta NA NEG

1727 CYB5R3 cytochrome b5 reductase 3 NA NEG

9939 RBM8A RNA binding motif protein 8A NA NEG

79027 ZNF655 zinc finger protein 655 NA NEG

FYVE, RhoGEF and PH domain

89846 FGD3 containing 3 NA NEG

4154 MBNL1 muscleblind-like (Drosophila) NA NEG

57018 CCNL1 Cyclin LI NA NEG

3094 HINT1 histidine triad nucleotide binding protein 1 NA NEG

1974 EIF4A2 eukaryotic translation initiation factor 4A2 NA NEG

1452 CSNK1A1 Casein kinase 1 , alpha 1 NA NEG

26036 ZNF451 zinc finger protein 451 NA NEG

27436 EML4 Echinoderm microtubule associated NA NEG protein like 4

56987 BBX bobby sox homolog (Drosophila) NA NEG protein phosphatase 2, regulatory subunit

5527 PPP2R5C B', gamma NA NEG

Serine/threonine kinase receptor

11171 STRAP associated protein NA NEG

7798 LUZP1 leucine zipper protein 1 NA NEG intraflagellar transport 80 homolog

57560 IFT80 (Chlamydomonas) NA NEG

255394 TCP11L2 t-complex 11 (mouse)-like 2 NA NEG

25939 SAMHD1 SAM domain and HD domain 1 NA NEG

401261 FLJ38717 FLJ38717 protein NA NEG

81855 SFXN3 Sideroflexin 3 NA NEG pyridoxal-dependent decarboxylase

23042 PDXDC1 domain containing 1 NA NEG

26058 GIGYF2 GPvBlO interacting GYF protein 2 NA NEG

285331 CCDC66 coiled-coil domain containing 66 NA NEG

26986 PABPC1 Poly(A) binding protein, cytoplasmic 1 NA NEG myeloid/lymphoid or mixed-lineage

leukemia (trithorax homolog, Drosophila);

8028 MLLT10 translocated to, 10 NA NEG

57724 KIAA1632 KIAA1632 NA NEG

256021 LOC256021 hypothetical protein LOC256021 NA NEG

375 ARF 1 ADP-ribosylation factor 1 NA NEG

960 CD44 CD44 molecule (Indian blood group) NA NEG288656 LOC100288656 Similar to hCG2003116 NA NEG

64145 ZFYVE20 zinc finger, FYVE domain containing 20 NA NEG

196264 MPZL3 myelin protein zero-like 3 NA NEG

DnaJ (Hsp40) homolog, subfamily B,

3337 DNAJB1 member 1 NA NEG

6711 SPTBN1 spectrin, beta, non-erythrocytic 1 NA NEG

57062 DDX24 DEAD (Asp-Glu-Ala-Asp) box NA NEG polypeptide 24

protein tyrosine phosphatase type IVA,

7803 PTP4A1 member 1 NA NEG

4926 NUMA1 nuclear mitotic apparatus protein 1 NA NEG

Sjogren syndrome antigen B (autoantigen

6741 SSB La) NA NEG

ATPase, Na+/K+ transporting, beta 1

481 ATP1B1 polypeptide NA NEG

5869 RAB5B RAB5B, member RAS oncogene family NA NEG

3725 JUN jun oncogene NA NEG

3725 JUN jun oncogene NA NEG ariadne homolog, ubiquitin-conjugating

enzyme E2 binding protein, 1

25820 ARIH1 (Drosophila) NA NEG protein phosphatase 1 , regulatory

23645 PPP1R15A (inhibitor) subunit 15A NA NEG

PRP4 pre-mRNA processing factor 4

8899 PRPF4B homolog B (yeast) NA NEG calcium homeostasis endoplasmic

10523 CHERP reticulum protein NA NEG

PRP3 pre-mRNA processing factor 3

9129 PRPF3 homolog (S. cerevisiae) NA NEG

23219 FBX028 F-box protein 28 NA NEG serpin peptidase inhibitor, clade F (alpha- 2 antiplasmin, pigment epithelium derived

5176 SERPINF1 factor), member 1 NA NEG

11333 PDAP1 PDGFA associated protein 1 NA NEG

28951 TRIB2 tribbles homolog 2 (Drosophila) NA NEG sparc/osteonectin, cwcv and kazal-like

9806 SPOCK2 domains proteoglycan (testican) 2 NA NEG aminoacyl tRNA synthetase complex-

9255 AIMP1 interacting multifunctional protein 1 NA NEG 8204 NRIP1 nuclear receptor interacting protein 1 NA NEG

9282 MED 14 mediator complex subunit 14 NA NEG

5257 PHKB phosphorylase kinase, beta NA NEG splicing factor, arginine/serine-rich 8

(suppressor-of-white-apricot homolog,

6433 SFRS8 Drosophila) NA NEG dual-specificity tyrosine-(Y)-

8445 DYRK2 phosphorylation regulated kinase 2 NA NEG

7390 UROS uroporphyrinogen III synthase NA NEG solute carrier family 11 (proton-coupled

4891 SLC11A2 divalent metal ion transporters), member 2 NA NEG

7019 TFAM transcription factor A, mitochondrial NA NEG

1633 DCK deoxycytidine kinase NA NEG

7050 TGIF1 TGFB-induced factor homeobox 1 NA NEG inositol polyphosphate-5 -phosphatase,

3635 INPP5D 145kDa NA NEG adaptor-related protein complex 1 , gamma

164 AP1G1 1 subunit NA NEG

9882 TBC1D4 TBC 1 domain family, member 4 NA NEG

UDP-N-acetyl-alpha-D- galactosamine:polypeptide N- acetylgalactosaminyltransferase 3

2591 GALNT3 (GalNAc-T3) NA NEG

5469 MED1 mediator complex subunit 1 NA NEG

24149 ZNF318 zinc finger protein 318 NA NEG solute carrier family 7 (cationic amino

9057 SLC7A6 acid transporter, y+ system), member 6 NA NEG dynein, cytoplasmic 1 , light intermediate

1783 DYNC1LI2 chain 2 NA NEG

3480 IGF1R insulin-like growth factor 1 receptor NA NEG

3727 JUND jun D proto-oncogene NA NEG

3727 JUND jun D proto-oncogene NA NEG 23635 SSBP2 single-stranded DNA binding protein 2 NA NEG

RABl 1 family interacting protein 2 (class

22841 RAB11FIP2 I) NA NEG

4907 NT5E 5 '-nucleotidase, ecto (CD73) NA NEG

9749 PHACTR2 phosphatase and actin regulator 2 NA NEG topoisomerase I binding, arginine/serine-

10210 TOPORS rich NA NEG solute carrier family 5 (sodium-dependent

8884 SLC5A6 vitamin transporter), member 6 NA NEG purinergic receptor P2X, ligand-gated ion

5025 P2RX4 channel, 4 NA NEG

7748 ZNF195 zinc finger protein 195 NA NEG chromodomain helicase DNA binding

1105 CHD1 protein 1 NA NEG phorbol- 12-myristate- 13 -acetate-induced

5366 PMAIP1 protein 1 NA NEG phorbol- 12-myristate- 13 -acetate-induced

5366 PMAIP1 protein 1 NA NEG

7753 ZNF202 zinc finger protein 202 NA NEG

7188 TRAF5 TNF receptor-associated factor 5 NA NEG

DEAH (Asp-Glu-Ala-His) box

22907 DHX30 polypeptide 30 NA NEG

22863 KIAA0831 KIAA0831 NA NEG nuclear receptor subfamily 4, group A,

4929 NR4A2 member 2 NA NEG nuclear receptor subfamily 4, group A,

4929 NR4A2 member 2 NA NEG signal transducer and activator of

6777 STAT5B transcription 5B NA NEG phosphatidylinositol glycan anchor

5277 PIGA biosynthesis, class A NA NEG

5816 PVALB parvalbumin NA NEG 1386 ATF2 activating transcription factor 2 NA NEG

3241 HPCAL1 hippocalcin-like 1 NA NEG

64848 YTHDC2 YTH domain containing 2 NA NEG

10863 ADAM28 ADAM metallopeptidase domain 28 NA NEG

6431 SFRS6 splicing factor, arginine/serine-rich 6 NA NEG

5868 RAB5A RAB5A, member RAS oncogene family NA NEG

640 BLK B lymphoid tyrosine kinase NA NEG

GTPase activating protein (SH3 domain)

9908 G3BP2 binding protein 2 NA NEG

2272 FHIT fragile histidine triad gene NA NEG

9205 ZMYM5 zinc finger, MYM-type 5 NA NEG

10225 CD96 CD96 molecule NA NEG

116228 /// FAM36A /// family with sequence similarity 36,

3204 HOXA7 member A /// homeobox A7 NA NEG

11278 KLF12 Kruppel-like factor 12 NA NEG

2323 FLT3LG fms-related tyrosine kinase 3 ligand NA NEG

1831 TSC22D3 TSC22 domain family, member 3 NA NEG

10001 MED6 mediator complex subunit 6 NA NEG

O-linked N-acetylglucosamine (GlcNAc)

transferase (UDP-N- acetylglucosamine:polypeptide-N-

8473 OGT acetylglucosaminyl transferase) NA NEG

11273 ATXN2L ataxin 2-like NA NEG

7543 ZFX zinc finger protein, X-linked NA NEG

923 CD6 CD6 molecule NA NEG

SMT3 suppressor of mif two 3 homolog 1

7341 SUMOl (S. cerevisiae) NA NEG

5747 PTK2 PTK2 protein tyrosine kinase 2 NA NEG

GTPase activating protein (SH3 domain)

9908 G3BP2 binding protein 2 NA NEG

1316 KLF6 Kruppel-like factor 6 NA NEG

1316 KLF6 Kruppel-like factor 6 NA NEG 57035 Clorf63 chromosome 1 open reading frame 63 NA NEG

9776 KIAA0652 KIAA0652 NA NEG ubiquitin-conjugating enzyme E2G 2

7327 UBE2G2 (UBC7 homolog, yeast) NA NEG

CDC5 cell division cycle 5-like (S.

988 CDC5L pombe) NA NEG

6307 SC4MOL sterol-C4-methyl oxidase-like NA NEG

56339 METTL3 methyltransferase like 3 NA NEG polymerase (RNA) I polypeptide C,

9533 POLR1C 30kDa NA NEG

288 ANK3 ankyrin 3, node of Ranvier (ankyrin G) NA NEG

2625 GATA3 GATA binding protein 3 NA NEG

2625 GATA3 GATA binding protein 3 NA NEG chromobox homolog 5 (HP1 alpha

23468 CBX5 homolog, Drosophila) NA NEG

969 CD69 CD69 molecule NA NEG aminoacyl tRNA synthetase complex-

7965 AIMP2 interacting multifunctional protein 2 NA NEG

SMGl homolog, phosphatidylinositol 3-

23049 SMGl kinase-related kinase (C. elegans) NA NEG

6430 SFRS5 splicing factor, arginine/serine-rich 5 NA NEG

6434 TRA2B transformer 2 beta homolog (Drosophila) NA NEG

22902 RUFY3 RUN and FYVE domain containing 3 NA NEG

2841 GPR18 G protein-coupled receptor 18 NA NEG membrane-spanning 4-domains,

931 MS4A1 subfamily A, member 1 NA NEG

8339 HIST1H2BG histone cluster 1, H2bg NA NEG

2323 FLT3LG fms-related tyrosine kinase 3 ligand NA NEG

11168 PSIP1 PC4 and SFRS 1 interacting protein 1 NA NEG

CCR4-NOT transcription complex,

4850 CNOT4 subunit 4 NA NEG

3572 IL6ST interleukin 6 signal transducer (gpl30, NA NEG oncostatin M receptor)

1606 DGKA diacylglycerol kinase, alpha 80kDa NA NEG tumor necrosis factor receptor

8718 TNFRSF25 superfamily, member 25 NA NEG

84786 MGC12488 hypothetical protein MGC12488 NA NEG

8725 C19orf2 chromosome 19 open reading frame 2 NA NEG ribosomal protein S6 kinase, 70kDa,

6198 RPS6KB1 polypeptide 1 NA NEG

1316 KLF6 Kruppel-like factor 6 NA NEG

581 BAX BCL2-associated X protein NA NEG tumor necrosis factor receptor

8718 TNFRSF25 superfamily, member 25 NA NEG

923 CD6 CD6 molecule NA NEG

10147 SFRS14 splicing factor, arginine/serine-rich 14 NA NEG

DEAH (Asp-Glu-Ala-His) box

1660 DHX9 polypeptide 9 NA NEG ankyrin repeat and LEM domain

23141 ANKLE2 containing 2 NA NEG

171023 ASXL1 additional sex combs like 1 (Drosophila) NA NEG

204851 HIPK1 homeodomain interacting protein kinase 1 NA NEG

23157 SEPT6 septin 6 NA NEG

E74-like factor 1 (ets domain transcription

1997 ELF1 factor) NA NEG

DEAD (Asp-Glu-Ala-Asp) box

1654 DDX3X polypeptide 3, X- linked NA NEG

DEAD (Asp-Glu-Ala-Asp) box

1654 DDX3X polypeptide 3, X- linked NA NEG

23348 DOCK9 dedicator of cytokinesis 9 NA NEG v-akt murine thymoma viral oncogene

10000 AKT3 homolog 3 (protein kinase B, gamma) NA NEG

23301 EHBP1 EH domain binding protein 1 NA NEG

23301 EHBP1 EH domain binding protein 1 NA NEG calcium/ calmodulin-dependent protein

818 CAMK2G kinase II gamma NA NEG

23177 CEP68 centrosomal protein 68kDa NA NEG

26043 UBXN7 UBX domain protein 7 NA NEG growth arrest and DNA-damage-

90480 GADD45GIP1 inducible, gamma interacting protein 1 NA NEG

57476 GRAMD1B GRAM domain containing IB NA NEG

7337 UBE3A ubiquitin protein ligase E3 A NA NEG

139322 APOOL Apolipoprotein O-like NA NEG heterogeneous nuclear ribonucleoprotein

3187 HNR PH1 HI (H) NA NEG

5094 PCBP2 poly(rC) binding protein 2 NA NEG

58487 CREBZF CREB/ATF bZIP transcription factor NA NEG inositol polyphosphate-5 -phosphatase,

3633 INPP5B 75kDa NA NEG

55556 ENOSF1 enolase superfamily member 1 NA NEG splicing factor, arginine/serine-rich 7,

6432 SFRS7 35kDa NA NEG

56339 METTL3 methyltransferase like 3 NA NEG

5936 RBM4 RNA binding motif protein 4 NA NEG

8930 MBD4 methyl-CpG binding domain protein 4 NA NEG

57125 PLXDC1 plexin domain containing 1 NA NEG cell division cycle 42 (GTP binding

998 CDC42 protein, 25kDa) NA NEG inhibitor of kappa light polypeptide gene

9641 IKBKE enhancer in B-cells, kinase epsilon NA NEG

9063 PIAS2 protein inhibitor of activated STAT, 2 NA NEG

RAP2A, member of RAS oncogene

5911 /// RAP2A /// family /// RAP2B, member of RAS

5912 RAP2B oncogene family NA NEG

MYST histone acetyltransferase

23522 MYST4 (monocytic leukemia) 4 NA NEG 10776 ARPP19 cAMP -regulated phosphoprotein, 19kDa NA NEG

1195 CLK1 CDC-like kinase 1 NA NEG mitogen-activated protein kinase kinase

4214 MAP3K1 kinase 1 NA NEG heterogeneous nuclear ribonucleoprotein

4670 HNR PM M NA NEG

23029 RBM34 RNA binding motif protein 34 NA NEG family with sequence similarity 153,

202134 /// FAM153A /// member A /// family with sequence

285596 /// FAM153B /// similarity 153, member B /// family with

653316 FAM153C sequence similarity 153, member C NA NEG

53371 NUP54 nucleoporin 54kDa NA NEG

80342 TRAF3IP3 TRAF3 interacting protein 3 NA NEG

926 CD8B CD8b molecule NA NEG

11221 DUSP10 dual specificity phosphatase 10 NA NEG

9725 TMEM63A Transmembrane protein 63A NA NEG

10198 MPHOSPH9 M-phase phosphoprotein 9 NA NEG

9397 NMT2 N-myristoyltransferase 2 NA NEG

22904 SBN02 strawberry notch homolog 2 (Drosophila) NA NEG

8339 HIST1H2BG histone cluster 1, H2bg NA NEG

8450 CUL4B cullin 4B NA NEG

960 CD44 CD44 molecule (Indian blood group) NA NEG

5609 MAP2K7 mitogen-activated protein kinase kinase 7 NA NEG nuclear receptor subfamily 4, group A,

4929 NR4A2 member 2 NA NEG

5996 RGS1 regulator of G-protein signaling 1 NA NEG

3712 IVD isovaleryl-CoA dehydrogenase NA NEG nuclear receptor subfamily 4, group A,

8013 NR4A3 member 3 NA NEG transducin-like enhancer of split 4 (E(spl)

7091 TLE4 homolog, Drosophila) NA NEG

7430 EZR ezrin NA NEG 283755 /// HERC2P2 /// hect domain and RLD 2 pseudogene 2 ///

400322 /// HERC2P3 /// hect domain and RLD 2 pseudogene 3 ///

440248 LOC440248 hect domain and RLD 2 pseudogene NA NEG membrane-spanning 4-domains,

931 MS4A1 subfamily A, member 1 NA NEG peptidylprolyl isomerase A (cyclophilin

5478 PPIA A) NA NEG

10209 EIF1 eukaryotic translation initiation factor 1 NA NEG

51335 NGRN neugrin, neurite outgrowth associated NA NEG

2873 GPS 1 G protein pathway suppressor 1 NA NEG

51646 YPEL5 yippee-like 5 (Drosophila) NA NEG

55968 NSFL1C NSFL1 (p97) cofactor (p47) NA NEG

23469 PHF3 PHD finger protein 3 NA NEG

29105 C16orf80 chromosome 16 open reading frame 80 NA NEG

64061 TSPYL2 TSPY-like 2 NA NEG pyruvate dehyrogenase phosphatase

54704 PDP1 catalytic subunit 1 NA NEG ankyrin repeat and zinc finger domain

55139 ANKZF1 containing 1 NA NEG

DEAH (Asp-Glu-Ala-His) box

79665 DHX40 polypeptide 40 NA NEG

64864 RFX7 regulatory factor X, 7 NA NEG

1877 E4F1 E4F transcription factor 1 NA NEG

55127 HEATR1 HEAT repeat containing 1 NA NEG

79693 YRDC yrdC domain containing (E. coli) NA NEG potassium channel tetramerisation domain

54793 KCTD9 containing 9 NA NEG

78991 PCYOX1L prenylcysteine oxidase 1 like NA NEG zinc finger and BTB domain containing

65986 ZBTB10 10 NA NEG

79074 C2orf49 chromosome 2 open reading frame 49 NA NEG

80146 UXS1 UDP-glucuronate decarboxylase 1 NA NEG 9840 KIAA0748 KIAA0748 NA NEG

79667 FLJ13197 hypothetical FLJ13197 NA NEG

55106 SLFN12 schlafen family member 12 NA NEG signal transducing adaptor family member

26228 STAP1 1 NA NEG

55180 LINS1 lines homolog 1 (Drosophila) NA NEG echinoderm microtubule associated

27436 EML4 protein like 4 NA NEG

ATG16 autophagy related 16-like 1 (S.

55054 ATG16L1 cerevisiae) NA NEG

79368 FCRL2 Fc receptor-like 2 NA NEG

11221 DUSP10 dual specificity phosphatase 10 NA NEG mitogen-activated protein kinase kinase

10746 MAP3K2 kinase 2 NA NEG

Splicing factor proline/glutamine-rich

(polypyrimidine tract binding protein

6421 SFPQ associated) NA NEG

83752 LONP2 Lon peptidase 2, peroxisomal NA NEG heterogeneous nuclear ribonucleoprotein

3191 HNR PL L NA NEG heterogeneous nuclear ribonucleoprotein

3178 HNR PA1 Al NA NEG

1540 CYLD cylindromatosis (turban tumor syndrome) NA NEG

57466 SFRS15 splicing factor, arginine/serine-rich 15 NA NEG

51646 YPEL5 yippee-like 5 (Drosophila) NA NEG tankyrase, TRF1 -interacting ankyrin-

80351 TNKS2 related ADP-ribose polymerase 2 NA NEG

79693 YRDC yrdC domain containing (E. coli) NA NEG

64940 STAG3L4 stromal antigen 3 -like 4 NA NEG

51762 RAB8B RAB8B, member RAS oncogene family NA NEG

9530 BAG4 BCL2-associated athanogene 4 NA NEG

55024 BANK1 B-cell scaffold protein with ankyrin NA NEG repeats 1

9840 KIAA0748 KIAA0748 NA NEG

51429 SNX9 sorting nexin 9 NA NEG

51429 SNX9 sorting nexin 9 NA NEG family with sequence similarity 60,

58516 /// FAM60A /// member A /// similar to family with

728115 LOC728115 sequence similarity 60, member A NA NEG

117177 RAB3IP RAB3A interacting protein (rabin3) NA NEG

56882 CDC42SE1 CDC42 small effector 1 NA NEG catenin (cadherin-associated protein), beta

1499 CTNNB1 1, 88kDa NA NEG

442582 /// STAG3L1 /// stromal antigen 3 -like 1 /// stromal antigen

54441 STAG3L2 3-like 2 NA NEG

114224 PR02852 hypothetical protein PR02852 NA NEG

90799 CCDC45 Coiled-coil domain containing 45 NA NEG

27086 FOXP1 forkhead box PI NA NEG

5150 PDE7A phosphodiesterase 7A NA NEG metastasis associated lung

adenocarcinoma transcript 1 (non-protein

378938 MALAT1 coding) NA NEG

1316 KLF6 Kruppel-like factor 6 NA NEG

84248 FYTTD1 forty-two-three domain containing 1 NA NEG

Ral GTPase activating protein, beta

57148 RALGAPB subunit (non-catalytic) NA NEG

84128 WDR75 WD repeat domain 75 NA NEG

LIM domain containing preferred

4026 LPP translocation partner in lipoma NA NEG nuclear fragile X mental retardation

57532 NUFIP2 protein interacting protein 2 NA NEG

57602 USP36 ubiquitin specific peptidase 36 NA NEG regulation of nuclear pre-mRNA domain

58490 RPRD1B containing IB NA NEG biorientation of chromosomes in cell

91272 BOD1 division 1 NA NEG

51274 KLF3 Kruppel-like factor 3 (basic) NA NEG

92400 RBM18 RNA binding motif protein 18 NA NEG

3840 KPNA4 karyopherin alpha 4 (importin alpha 3) NA NEG

90809 TMEM55B transmembrane protein 55B NA NEG

57693 ZNF317 zinc finger protein 317 NA NEG

84320 ACBD6 acyl-CoA binding domain containing 6 NA NEG enhancer of polycomb homolog 1

80314 EPC1 (Drosophila) NA NEG euchromatic histone-lysine N-

79813 EHMT1 methyltransferase 1 NA NEG

64651 CSR P1 cysteine-serine-rich nuclear protein 1 NA NEG

Mdm4 p53 binding protein homolog

4194 MDM4 (mouse) NA NEG adaptor-related protein complex 1 , gamma

164 AP1G1 1 subunit NA NEG

146198 ZFP90 zinc finger protein 90 homolog (mouse) NA NEG

RPTOR independent companion of

253260 RICTOR MTOR, complex 2 NA NEG

6711 SPTBN1 spectrin, beta, non-erythrocytic 1 NA NEG family with sequence similarity 117,

150864 FAM117B member B NA NEG

51762 RAB8B RAB8B, member RAS oncogene family NA NEG

221830 TWISTNB TWIST neighbor NA NEG

64839 FBXL17 F-box and leucine-rich repeat protein 17 NA NEG

151195 CCNYL1 cyclin Y-like 1 NA NEG

Smith-Magenis syndrome chromosome

140775 SMCR8 region, candidate 8 NA NEG

84236 RHBDD1 rhomboid domain containing 1 NA NEG

205717 KIAA2018 KIAA2018 NA NEG

203522 DDX26B DEAD/H (Asp-Glu-Ala- Asp/His) box NA NEG polypeptide 26B

4950 OCLN occludin NA NEG

57559 STAMBPL1 STAM binding protein-like 1 NA NEG

196264 MPZL3 myelin protein zero-like 3 NA NEG

90390 MED30 mediator complex subunit 30 NA NEG

TAF15 RNA polymerase II, TATA box

binding protein (TBP)-associated factor,

8148 TAF15 68kDa NA NEG

64343 AZI2 5-azacytidine induced 2 NA NEG family with sequence similarity 85,

FAM85A /// member A /// family with sequence

FAM85B similarity 85, member B NA NEG myosin regulatory light chain interacting

29116 MYLIP protein NA NEG

25981 DNAH1 dynein, axonemal, heavy chain 1 NA NEG growth arrest-specific 5 (non-protein

60674 GAS 5 coding) NA NEG

85451 UNK unkempt homolog (Drosophila) NA NEG ligand dependent nuclear receptor

84458 LCOR corepressor NA NEG

124491 TMEM170A transmembrane protein 170A NA NEG membrane-spanning 4-domains,

931 MS4A1 subfamily A, member 1 NA NEG membrane-spanning 4-domains,

931 MS4A1 subfamily A, member 1 NA NEG myocardial infarction associated transcript

440823 MI AT (non-protein coding) NA NEG

152137 CCDC50 coiled-coil domain containing 50 NA NEG

960 CD44 CD44 molecule (Indian blood group) NA NEG ribosomal modification protein rimK-like

57494 PvIMKLB family member B NA NEG

8874 ARHGEF7 Rho guanine nucleotide exchange factor NA NEG (GEF) 7

255231 MCOLN2 mucolipin 2 NA NEG

51696 HECA headcase homolog (Drosophila) NA NEG

9685 CLINT 1 clathrin interactor 1 NA NEG

283663 LOC283663 hypothetical LOC283663 NA NEG spastic paraplegia 7 (pure and complicated

6687 SPG7 autosomal recessive) NA NEG family with sequence similarity 129,

199786 FAM129C member C NA NEG activating signal cointegrator 1 complex

10973 ASCC3 subunit 3 NA NEG

158830 CXorf65 chromosome X open reading frame 65 NA NEG

64776 Cl lorfl chromosome 11 open reading frame 1 NA NEG phosphatidylinositol glycan anchor

9487 PIGL biosynthesis, class L NA NEG

85476 GFM1 G elongation factor, mitochondrial 1 NA NEG

64419 MTMR14 myotubularin related protein 14 NA NEG

10782 ZNF274 zinc finger protein 274 NA NEG

9440 MED 17 mediator complex subunit 17 NA NEG echinoderm microtubule associated

27436 EML4 protein like 4 NA NEG

84196 USP48 ubiquitin specific peptidase 48 NA NEG

157574 /// FBX016 /// F-box protein 16 /// zinc finger protein

55893 ZNF395 395 NA NEG

92482 NCRNA00081 non-protein coding RNA 81 NA NEG

ATPase family, AAA domain containing

54454 ATAD2B 2B NA NEG glutaminyl-tRNA synthase (glutamine-

55278 QRSL1 hydrolyzing)-like 1 NA NEG

49854 ZNF295 zinc finger protein 295 NA NEG debranching enzyme homolog 1 (S.

51163 DBR1 cerevisiae) NA NEG 127933 UHMK1 U2AF homology motif (UHM) kinase 1 NA NEG

152137 CCDC50 coiled-coil domain containing 50 NA NEG protein phosphatase, Mg2+/Mn2+

152926 PPM IK dependent, IK NA NEG muskelin 1 , intracellular mediator

4289 MKLN1 containing kelch motifs NA NEG

6428 SFRS3 splicing factor, arginine/serine-rich 3 NA NEG

29121 CLEC2D C-type lectin domain family 2, member D NA NEG

57721 METTL14 methyltransferase like 14 NA NEG

22847 ZNF507 zinc finger protein 507 NA NEG

54813 KLHL28 kelch-like 28 (Drosophila) NA NEG

91526 ANKRD44 ankyrin repeat domain 44 NA NEG

2778 GNAS GNAS complex locus NA NEG major histocompatibility complex, class

3117 HLA-DQA1 II, DQ alpha 1 NA NEG

23392 KIAA0368 KIAA0368 NA NEG

55183 RIF1 RAPl interacting factor homo log (yeast) NA NEG

6232 RPS27 ribosomal protein S27 NA NEG protein phosphatase 1 , regulatory

5504 PPP1R2 (inhibitor) subunit 2 NA NEG134445 LOCI 00134445 hypothetical LOCI 00134445 NA NEG

Golgi-associated PDZ and coiled-coil

57120 GOPC motif containing NA NEG

Proteasome (prosome, macropain)

23198 PSME4 activator subunit 4 NA NEG

79609 C14orfl38 chromosome 14 open reading frame 138 NA NEG

374868 ATP9B ATPase, class II, type 9B NA NEG transient receptor potential cation channel,

54822 TRPM7 subfamily M, member 7 NA NEG

SIN3 homolog A, transcription regulator

25942 SIN3A (yeast) NA NEG

25942 SIN3A SIN3 homolog A, transcription regulator NA NEG (yeast)

SYS 1 Golgi-localized integral membrane

90196 SYS1 protein homo log (S. cerevisiae) NA NEG

100130733 IPO 11 /// importin 11 /// leucine rich repeat

/// 51194 LR C70 containing 70 NA NEG

Leucine rich repeat (in FLU) interacting

9208 LRRFIP1 protein 1 NA NEG

401320 LOC401320 hypothetical LOC401320 NA NEG

80264 ZNF430 zinc finger protein 430 NA NEG

23200 ATP11B ATPase, class VI, type 1 IB NA NEG

374920 C19orf68 chromosome 19 open reading frame 68 NA NEG

2971 GTF3A general transcription factor IIIA NA NEG

UDP-Gal:betaGlcNAc beta 1,4-

2683 B4GALT1 galactosyltransferase, polypeptide 1 NA NEG

Cell division cycle and apoptosis regulator

55749 CCAR1 1 NA NEG ubiquitin-conjugating enzyme E2B

7320 UBE2B (RAD6 homolog) NA NEG

10207 INADL InaD-like (Drosophila) NA NEG transmembrane emp24 protein transport

50999 TMED5 domain containing 5 NA NEG

Family with sequence similarity 120 A

158293 FAM120AOS opposite strand NA NEG signaling lymphocytic activation molecule

6504 SLAMF1 family member 1 NA NEG

10432 RBM14 R A binding motif protein 14 NA NEG

28984 C13orfl5 Chromosome 13 open reading frame 15 NA NEG kelch repeat and BTB (POZ) domain

84541 KBTBD8 containing 8 NA NEG

80342 TRAF3IP3 TRAF3 interacting protein 3 NA NEG ubiquitin-conjugating enzyme E2D 3

7323 UBE2D3 (UBC4/5 homolog, yeast) NA NEG 9818 NUPL1 nucleoporin like 1 NA NEG

57018 CCNL1 cyclin LI NA NEG calmodulin 1 (phosphorylase kinase,

801 CALM1 delta) NA NEG

730051 ZNF814 zinc finger protein 814 NA NEG

25831 HECTD1 HECT domain containing 1 NA NEG zinc finger and BTB domain containing

27107 ZBTB11 11 NA NEG

DIS3 mitotic control homolog (S.

129563 DIS3L2 cerevisiae)-like 2 NA NEG

RAN binding protein 2 /// RANBP2-like

and GRIP domain containing 1 ///

285190 /// RANBP2 /// RANBP2-like and GRIP domain

400966 /// RGPD1 /// containing 2 /// RANBP2-like and GRIP

5903 /// RGPD2 /// domain containing 3 /// RANBP2-like and

653489 /// RGPD3 /// GRIP domain containing 4 /// RANBP2- 727851 /// RGPD4 /// like and GRIP domain containing 5 ///

729540 /// RGPD5 /// RANBP2-like and GRIP domain

729857 /// RGPD6 /// containing 6 /// RANBP2-like and GRIP

84220 RGPD8 domain containing 8 NA NEG

56902 PNOl partner of NOB 1 homolog (S. cerevisiae) NA NEG

23420 /// NOMOl ///

283820 /// NOM02 /// NODAL modulator 1 /// NODAL

408050 NOM03 modulator 2 /// NODAL modulator 3 NA NEG signal transducer and activator of

transcription 3 (acute-phase response

6774 STAT3 factor) NA NEG

115350 FCRLl Fc receptor-like 1 NA NEG interleukin 28 receptor, alpha (interferon,

163702 IL28RA lambda receptor) NA NEG

54205 CYCS cytochrome c, somatic NA NEG

9749 PHACTR2 phosphatase and actin regulator 2 NA NEG Proteasome (prosome, macropain)

5695 PSMB7 subunit, beta type, 7 NA NEG

Sfil homolog, spindle assembly

9814 SFI1 associated (yeast) NA NEG protein phosphatase 1 , regulatory

23645 PPP1R15A (inhibitor) subunit 15A NA NEG

1540 CYLD cylindromatosis (turban tumor syndrome) NA NEG

65268 WNK2 WNK lysine deficient protein kinase 2 NA POS

343099 CCDC18 Coiled-coil domain containing 18 NA POS

25981 DNAH1 dynein, axonemal, heavy chain 1 NA POS heat shock protein 90kDa beta (Grp94),

7184 HSP90B1 member 1 NA POS

9761 MLEC malectin NA POS

5034 P4HB prolyl 4-hydroxylase, beta polypeptide NA POS

KDEL (Lys-Asp-Glu-Leu) endoplasmic

11014 KDELR2 reticulum protein retention receptor 2 NA POS

50 AC02 aconitase 2, mitochondrial NA POS

10525 HYOU1 hypoxia up-regulated 1 NA POS

23022 PALLD palladin, cytoskeletal associated protein NA POS signal recognition particle receptor

6734 SRPR (docking protein) NA POS

CD59 molecule, complement regulatory

966 CD59 protein NA POS

6184 RPN1 ribophorin I NA POS phosphatidylinositol transfer protein,

5306 PITPNA alpha NA POS signal peptidase complex subunit 2

homolog pseudogene /// signal peptidase

653566 /// LOC653566 /// complex subunit 2 homolog (S.

9789 SPCS2 cerevisiae) NA POS

653566 /// LOC653566 /// signal peptidase complex subunit 2

9789 SPCS2 homolog pseudogene /// signal peptidase NA POS complex subunit 2 homolog (S.

cerevisiae)

ubiquinol-cytochrome c reductase,

27089 UQCRQ complex III subunit VII, 9.5kDa NA POS

8508 NIPSNAP1 nipsnap homolog 1 (C. elegans) NA POS

6745 SSR1 signal sequence receptor, alpha NA POS

6732 SRPK1 SFRS protein kinase 1 NA POS calcium homeostasis endoplasmic

10523 CHERP reticulum protein NA POS

9871 SEC24D SEC24 family, member D (S. cerevisiae) NA POS

10237 SLC35B1 solute carrier family 35, member Bl NA POS

7298 TYMS thymidylate synthetase NA POS mesencephalic astrocyte-derived

7873 MANF neurotrophic factor NA POS

11065 UBE2C ubiquitin-conjugating enzyme E2C NA POS ras homolog gene family, member G (rho

391 RHOG G) NA POS gamma-glutamyl hydrolase (conjugase,

8836 GGH folylpolygammaglutamyl hydrolase) NA POS

NADH dehydrogenase (ubiquinone) 1

4712 NDUFB6 beta subcomplex, 6, 17kDa NA POS

11130 ZWINT ZW10 interactor NA POS phospho lipase C, gamma 2

5336 PLCG2 (phosphatidylinositol-specific) NA POS

5450 POU2AF1 POU class 2 associating factor 1 NA POS

952 CD38 CD38 molecule NA POS

5368 PNOC prepronociceptin NA POS

1178 CLC Charcot-Leyden crystal protein NA POS apolipoprotein B mRNA editing enzyme,

9582 APOBEC3B catalytic polypeptide-like 3B NA POS tumor necrosis factor receptor

608 TNFRSF17 superfamily, member 17 NA POS 3577 CXCR1 chemokine (C-X-C motif) receptor 1 NA POS protein disulfide isomerase family A,

10130 PDIA6 member 6 NA POS ectonucleoside triphosphate

953 ENTPD1 diphosphohydrolase 1 NA POS

25840 METTL7A methyltransferase like 7A NA POS

4121 MAN1A1 mannosidase, alpha, class 1A, member 1 NA POS protein disulfide isomerase family A,

10130 PDIA6 member 6 NA POS protein disulfide isomerase family A,

9601 PDIA4 member 4 NA POS

6185 RPN2 ribophorin II NA POS

ATPase, Na+/K+ transporting, beta 3

483 ATP1B3 polypeptide NA POS

3535 abParts abParts NA POS glycoprotein lb (platelet), beta

2812 GP1BB polypeptide NA POS

6241 RRM2 ribonucleotide reductase M2 NA POS

10211 FLOT1 flotillin 1 NA POS

3705 ITPK1 inositol 1,3,4-triphosphate 5/6 kinase NA POS protein disulfide isomerase family A,

9601 PDIA4 member 4 NA POS immunoglobulin heavy locus ///

immunoglobulin heavy constant alpha 1

28396 /// IGH@ /// /// immunoglobulin heavy constant alpha

3492 /// IGHA1 /// 2 (A2m marker) /// immunoglobulin

3493 /// IGHA2 /// heavy constant gamma 1 (Glm marker) ///

3494 /// IGHG1 /// immunoglobulin heavy constant gamma 3

3500 /// IGHG3 /// (G3m marker) /// immunoglobulin heavy

3502 /// IGHM /// constant mu /// immunoglobulin heavy

3507 IGHV4-31 variable 4-31 NA POS

100290146 abParts abParts NA POS ///

100290528

/// 28396 ///

28442 ///

3492 ///

3493 ///

3495 ///

3500 ///

3502 ///

3503 ///

3507

100290146

///

100290528

/// 28396 ///

3492 ///

3493 ///

3494 ///

3495 ///

3500 ///

3502 ///

3503 ///

3507 abParts abParts NA POS

100290320

///

100291190

/// 28396 ///

3492 ///

3493 ///

3494 ///

3495 ///

3500 ///

3502 /// abParts abParts NA POS 3507

28875 ///

3514 ///

50802 abParts abParts NA POS

Transcribed

Locus abParts abParts NA POS

28831 abParts abParts NA POS

100126583

///

100290320

///

100291190

/// 28396 ///

3492 ///

3493 ///

3494 ///

3495 ///

3500 ///

3501 ///

3502 ///

3507 abParts abParts NA POS

28831 abParts abParts NA POS

28912 IGKV3-20 Immunoglobulin kappa variable 3-20 NA POS

857 CAV1 caveolin 1 , caveolae protein, 22kDa NA POS proteasome (prosome, macropain) 26S

10213 PSMD14 subunit, non-ATPase, 14 NA POS cAMP responsive element binding protein

64764 CREB3L2 3-like 2 NA POS

23065 KIAA0090 KIAA0090 NA POS solute carrier family 30 (zinc transporter),

7779 SLC30A1 member 1 NA POS

137886 UBXN2B UBX domain protein 2B NA POS 4781 NFIB nuclear factor I/B NA POS adaptor-related protein complex 3, sigma

10239 AP3S2 2 subunit NA POS

890 CCNA2 cyclin A2 NA POS serine hydroxymethyltransferase 2

6472 SHMT2 (mitochondrial) NA POS opioid binding protein/cell adhesion

4978 OPCML molecule-like NA POS

9236 CCPG1 cell cycle progression 1 NA POS

3535 IGL@ Immunoglobulin lambda locus NA POS

100290059

///

100292999

/// 3495 abParts abParts NA POS

28778 abParts abParts NA POS

100290481 CYAT1 /// cyclosporin A transporter 1 ///

/// 28823 IGLV1-44 immunoglobulin lambda variable 1-44 NA POS immunoglobulin lambda constant 1 (Meg

100423062 IGLCl /// marker) /// immunoglobulin lambda-like

/// 28793 /// IGLL5 /// polypeptide 5 /// immunoglobulin lambda

28799 /// IGLV3-16 /// variable 3-16 /// immunoglobulin lambda

3537 IGLV3-25 variable 3-25 NA POS

28823 abParts abParts NA POS

Catenin (cadherin-associated protein),

1495 CTNNA1 alpha 1, 102kDa NA POS immunoglobulin lambda-like polypeptide

1 /// immunoglobulin lambda-like

3543 /// IGLL1 /// polypeptide 3 /// glucuronidase,

91316 /// IGLL3 /// beta/immunoglobulin lambda-like

91353 LOC91316 polypeptide 1 pseudogene NA POS

3507 ///

652494 abParts abParts NA POS 6955 TRA@ T cell receptor alpha locus NA POS

28902 abParts abParts NA POS

652493 abParts abParts NA POS

100290557 IGLV1-44 /// immunoglobulin lambda variable 1-44 ///

/// 28823 LOC100290557 similar to hCG91935 NA POS heat shock protein 90kDa beta (Grp94),

7184 HSP90B1 member 1 NA POS

3507 abParts abParts NA POS

100133862 IGHG1 /// immunoglobulin heavy constant gamma 1

/// 3500 /// IGHM /// (Glm marker) /// immunoglobulin heavy

3507 LOC100133862 constant mu /// similar to hCG1773549 NA POS

100290293

/// 3493 ///

3500 ///

3507 abParts abParts NA POS

100290320

///

100291190

/// 28396 ///

3493 ///

3495 ///

3500 ///

3502 ///

3507 abParts abParts NA POS immunoglobulin kappa locus ///

3514 /// immunoglobulin kappa constant /// similar

50802 /// IGK@ /// IGKC to Ig kappa chain V-I region HK102

652493 /// /// LOC652493 precursor /// similar to Ig kappa chain V-I

652694 /// LOC652694 region HK102 precursor NA POS

100293440 abParts abParts NA POS

3514 ///

50802 ///

652493 abParts abParts NA POS 100290557 abParts abParts NA POS immunoglobulin heavy locus ///

immunoglobulin heavy constant alpha 1

/// immunoglobulin heavy constant alpha

100126583 IGH@ /// 2 (A2m marker) /// immunoglobulin

/// 3492 /// IGHA1 /// heavy constant delta /// immunoglobulin

3493 /// IGHA2 /// heavy constant gamma 1 (Glm marker) ///

3494 /// IGHD /// immunoglobulin heavy constant gamma 3

3495 /// IGHG1 /// (G3m marker) /// immunoglobulin heavy

3500 /// IGHG3 /// constant gamma 4 (G4m marker) ///

3502 /// IGHG4 /// immunoglobulin heavy constant mu ///

3503 /// IGHM /// hypothetical LOCI 00126583 /// similar to

3507 /// LOC100126583 Ig heavy chain V-II region ARH-77

652128 /// LOC652128 precursor NA POS

100290557

/// 28823 abParts abParts NA POS

861 RUNX1 runt-related transcription factor 1 NA POS

100130100 LOC100130100

/// /// similar to hCG26659 /// similar to

100291464 LOC100291464 hCG26659 NA POS

100287723 LOCI 00287723 similar to Ig kappa chain NA POS ubiquitin-conjugating enzyme E2, Jl

51465 UBE2J1 (UBC6 homolog, yeast) NA POS ubiquitin-conjugating enzyme E2, Jl

51465 UBE2J1 (UBC6 homolog, yeast) NA POS

51065 RPS27L ribosomal protein S27-like NA POS

8566 PDXK pyridoxal (pyridoxine, vitamin B6) kinase NA POS

746 Cl lorflO chromosome 11 open reading frame 10 NA POS

55365 TMEM176A transmembrane protein 176A NA POS translocase of inner mitochondrial

26521 TIMM8B membrane 8 homolog B (yeast) NA POS

79174 CRELD2 cysteine-rich with EGF-like domains 2 NA POS calcium regulated heat stable protein 1 ,

23589 CARHSP1 24kDa NA POS small nuclear ribonucleoprotein 25kDa

79622 SNR P25 (U11/U12) NA POS

54998 AURKAIP1 aurora kinase A interacting protein 1 NA POS

79694 MANEA mannosidase, endo-alpha NA POS

56946 Cl lorOO chromosome 11 open reading frame 30 NA POS

57823 SLAMF7 SLAM family member 7 NA POS

55840 EAF2 ELL associated factor 2 NA POS

63910 SLC17A9 solute carrier family 17, member 9 NA POS

79906 MORN1 MORN repeat containing 1 NA POS

79770 TXNDC15 thioredoxin domain containing 15 NA POS

64062 RBM26 RNA binding motif protein 26 NA POS muted homolog (mouse) /// thioredoxin

63915 /// MUTED /// domain containing 5 (endoplasmic

81567 TXNDC5 reticulum) NA POS

51237 MGC29506 plasma cell-induced ER protein 1 NA POS

56005 C19orfl0 chromosome 19 open reading frame 10 NA POS

4121 MAN1A1 Mannosidase, alpha, class 1A, member 1 NA POS

3069 HDLBP high density lipoprotein binding protein NA POS

23420 /// NOMOl ///

283820 /// NOM02 /// NODAL modulator 1 /// NODAL

408050 NOM03 modulator 2 /// NODAL modulator 3 NA POS

55858 TMEM165 transmembrane protein 165 NA POS gem (nuclear organelle) associated protein

79760 GEMIN7 7 NA POS

81037 CLPTM1L CLPTMl-like NA POS

51258 MRPL51 mitochondrial ribosomal protein L51 NA POS

90701 SEC11C SEC 11 homolog C (S. cerevisiae) NA POS

727751 /// LOC727751 /// hypothetical LOC727751 /// similar to cis- 727849 /// LOC727849 /// Golgi matrix protein GM130 ///

80154 LOC80154 hypothetical LOC80154 NA POS 51237 MGC29506 plasma cell-induced ER protein 1 NA POS phosphatidic acid phosphatase type 2

84513 PPAPDC1B domain containing IB NA POS

23446 SLC44A1 solute carrier family 44, member 1 NA POS

23446 SLC44A1 solute carrier family 44, member 1 NA POS

27248 ERLEC1 endoplasmic reticulum lectin 1 NA POS

23184 MESDC2 mesoderm development candidate 2 NA POS

90231 KIAA2013 KIAA2013 NA POS tumor protein p53 inducible nuclear

94241 TP53INP1 protein 1 NA POS

2140 EYA3 eyes absent homolog 3 (Drosophila) NA POS

126321 C19orf28 chromosome 19 open reading frame 28 NA POS

56977 STOX2 storkhead box 2 NA POS coiled-coil-helix-coiled-coil-helix domain

118487 CHCHD1 containing 1 NA POS

81037 CLPTM1L CLPTMl-like NA POS

387103 CENPW centromere protein W NA POS low density lipoprotein receptor-related

26020 LRP10 protein 10 NA POS

790955 Cl lorf83 chromosome 11 open reading frame 83 NA POS

UDP-glucose glycoprotein

56886 UGGT1 glucosyltransferase 1 NA POS

100293277 IGLCl /// immunoglobulin lambda constant 1 (Meg

/// 3537 LOCI 00293277 marker) /// similar to hCG2040021 NA POS

100290481 abParts abParts NA POS

7678 ZNF124 zinc finger protein 124 NA POS

28823 abParts abParts NA POS

55924 Clorfl83 chromosome 1 open reading frame 183 NA POS

3310 HSPA6 heat shock 70kDa protein 6 (HSP70B ^* ) POS NA

5594 MAPK1 mitogen-activated protein kinase 1 POS NA

GRB2-binding adaptor protein,

202309 GAPT transmembrane POS NA 151306 GPBAR1 G protein-coupled bile acid receptor 1 POS NA

89790 /// SIGLEC10 /// sialic acid binding Ig-like lectin 10 ///

89858 SIGLEC12 sialic acid binding Ig-like lectin 12 POS NA colony stimulating factor 3 receptor

1441 CSF3R (granulocyte) POS NA

729991 LOC729991 hypothetical protein LOC729991 POS NA

79650 C16orf57 chromosome 16 open reading frame 57 POS NA

UEV and lactate/malate dehyrogenase

55293 UEVLD domains POS NA

7439 BEST1 bestrophin 1 POS NA caspase recruitment domain family,

22900 CARD 8 member 8 POS NA

10326 SIRPB1 signal-regulatory protein beta 1 POS NA isocitrate dehydrogenase 1 (NADP+),

3417 IDH1 soluble POS NA

4668 NAGA N-acetylgalactosaminidase, alpha- POS NA

284759 SIRPB2 signal-regulatory protein beta 2 POS NA

5256 PHKA2 phosphorylase kinase, alpha 2 (liver) POS NA

285708 LOC285708 hypothetical protein LOC285708 POS NA

FYVE, RhoGEF and PH domain

221472 FGD2 containing 2 POS NA protein phosphatase 1 , regulatory

6992 PPP1R11 (inhibitor) subunit 11 POS NA

140890 SFRS12 splicing factor, arginine/serine-rich 12 POS NA peroxisome proliferator-activated receptor

10891 PPARGC1A gamma, coactivator 1 alpha POS NA

826 CAPNS1 calpain, small subunit 1 POS NA protein kinase, cAMP-dependent,

regulatory, type I, alpha (tissue specific

5573 PRKAR1A extinguisher 1) POS NA protein kinase, cAMP-dependent,

5573 PRKAR1A regulatory, type I, alpha (tissue specific POS NA extinguisher 1)

CAP, adenylate cyclase-associated protein

10487 CAP1 1 (yeast) POS NA

3069 HDLBP high density lipoprotein binding protein POS NA

6282 S100A11 SI 00 calcium binding protein Al 1 POS NA

2896 CRN granulin POS NA

2934 GSN gelsolin POS NA

3098 HK1 hexokinase 1 POS NA

KDEL (Lys-Asp-Glu-Leu) endoplasmic

11014 KDELR2 reticulum protein retention receptor 2 POS NA

377 ARF3 ADP-ribosylation factor 3 POS NA

1808 DPYSL2 dihydropyrimidinase-like 2 POS NA

1509 CTSD cathepsin D POS NA low density lipoprotein receptor-related

4035 LRP1 protein 1 POS NA

7167 TPI1 triosephosphate isomerase 1 POS NA

10768 AHCYL1 adenosylhomocysteinase-like 1 POS NA

10768 AHCYL1 adenosylhomocysteinase-like 1 POS NA mannose-6-phosphate receptor (cation

4074 M6PR dependent) POS NA

KDEL (Lys-Asp-Glu-Leu) endoplasmic

10945 KDELRl reticulum protein retention receptor 1 POS NA

1211 CLTA clathrin, light chain A POS NA ubiquitin-like modifier activating enzyme

7317 UBA1 1 POS NA

ARP3 actin-related protein 3 homolog

10096 ACTR3 (yeast) POS NA

3728 JUP junction plakoglobin POS NA

ATPase, H+ transporting, lysosomal

526 ATP6V1B2 56/58kDa, VI subunit B2 POS NA

47 ACLY ATP citrate lyase POS NA

3417 IDH1 isocitrate dehydrogenase 1 (NADP+), POS NA soluble

1476 CSTB cystatin B (stefin B) POS NA

1488 CTBP2 C-terminal binding protein 2 POS NA

3611 ILK integrin-linked kinase POS NA heterogeneous nuclear ribonucleoprotein

3182 HNR PAB A/B POS NA oxoglutarate (alpha-ketoglutarate)

4967 OGDH dehydrogenase (lipoamide) POS NA

327 APEH N-acylaminoacyl -peptide hydrolase POS NA interferon induced transmembrane protein

10581 IFITM2 2 (1-8D) POS NA

79602 ADIPOR2 adiponectin receptor 2 POS NA protein phosphatase 2, catalytic subunit,

5516 PPP2CB beta isozyme POS NA

4259 MGST3 microsomal glutathione S -transferase 3 POS NA

10437 IFI30 interferon, gamma-inducible protein 30 POS NA

5547 PRCP prolylcarboxypeptidase (angiotensinase C) POS NA protein phosphatase 1 , regulatory

6992 PPPlRl l (inhibitor) subunit 11 POS NA

10483 SEC23B Sec23 homolog B (S. cerevisiae) POS NA structural maintenance of chromosomes

8243 SMC1A 1A POS NA interferon gamma receptor 2 (interferon

3460 IFNGR2 gamma transducer 1) POS NA protein kinase C and casein kinase

11252 PACSIN2 substrate in neurons 2 POS NA

6642 SNX1 sorting nexin 1 POS NA

9690 UBE3C ubiquitin protein ligase E3C POS NA family with sequence similarity 127,

8933 FAM127A member A POS NA capping protein (actin filament), gelsolin-

822 CAPG like POS NA aldo-keto reductase family 1, member Al

10327 AK 1A1 (aldehyde reductase) POS NA actin related protein 2/3 complex, subunit

10095 ARPC1B IB, 41kDa POS NA

9897 KIAA0196 KIAA0196 POS NA

5184 PEPD peptidase D POS NA

1445 CSK c-src tyrosine kinase POS NA

1861 TORI A torsin family 1 , member A (torsin A) POS NA adaptor-related protein complex 3, sigma

10239 AP3S2 2 subunit POS NA

6256 PvXPvA retinoid X receptor, alpha POS NA

10014 HDAC5 histone deacetylase 5 POS NA tumor necrosis factor, alpha-induced

7127 TNFAIP2 protein 2 POS NA

5580 PR CD protein kinase C, delta POS NA mitogen-activated protein kinase-activated

7867 MAPKAPK3 protein kinase 3 POS NA protein kinase, cAMP-dependent,

5566 PRKACA catalytic, alpha POS NA

54838 C10orf26 chromosome 10 open reading frame 26 POS NA

2548 GAA glucosidase, alpha; acid POS NA serpin peptidase inhibitor, clade A (alpha-

5265 SERPINA1 1 antiproteinase, antitrypsin), member 1 POS NA

10928 RALBP1 ralA binding protein 1 POS NA

22918 CD93 CD93 molecule POS NA

290 ANPEP alanyl (membrane) aminopeptidase POS NA

140885 SIRPA signal-regulatory protein alpha POS NA

4668 NAGA N-acetylgalactosaminidase, alpha- POS NA

3145 HMBS hydroxymethylbilane synthase POS NA

3920 LAMP2 lysosomal-associated membrane protein 2 POS NA carbohydrate (N-acetylgalactosamine 4-

51363 CHST15 sulfate 6-0) sulfotransferase 15 POS NA 3613 IMPA2 inositol(myo)-l(or 4)-monophosphatase 2 POS NA

9830 TRIM 14 tripartite motif-containing 14 POS NA

Ras association (RalGDS/AF-6) domain

9770 RASSF2 family member 2 POS NA

3660 IRF2 interferon regulatory factor 2 POS NA ribosomal protein S6 kinase, 90kDa,

6195 RPS6KA1 polypeptide 1 POS NA damage-specific DNA binding protein 2,

1643 DDB2 48kDa POS NA

963 CD53 CD53 molecule POS NA

5663 PSEN1 presenilin 1 POS NA

10654 PMVK phosphomevalonate kinase POS NA

1130 LYST lysosomal trafficking regulator POS NA

920 CD4 CD4 molecule POS NA family with sequence similarity 193,

8603 FAM193A member A POS NA sulfotransferase family, cytosolic, 1A,

6817 SULT1A1 phenol-preferring, member 1 POS NA

10311 DSCR3 Down syndrome critical region gene 3 POS NA

6905 TBCE tubulin folding cofactor E POS NA

113 ADCY7 adenylate cyclase 7 POS NA grancalcin, EF-hand calcium binding

25801 GCA protein POS NA

4835 NQ02 NAD(P)H dehydrogenase, quinone 2 POS NA haloacid dehalogenase-like hydrolase

8226 HDHD1A domain containing 1 A POS NA ectonucleoside triphosphate

9583 ENTPD4 diphosphohydrolase 4 POS NA

T-cell, immune regulator 1 , ATPase, H+

10312 TCIRG1 transporting, lysosomal V0 subunit A3 POS NA

4258 MGST2 microsomal glutathione S -transferase 2 POS NA

5481 PPID peptidylprolyl isomerase D POS NA interferon (alpha, beta and omega)

3454 IFNAR1 receptor 1 POS NA vitamin D (1,25- dihydroxyvitamin D3)

7421 VDR receptor POS NA

9764 KIAA0513 KIAA0513 POS NA

11237 RNF24 ring finger protein 24 POS NA regulator of chromosome condensation

(RCC1) and BTB (POZ) domain

1102 RCBTB2 containing protein 2 POS NA

10875 FGL2 fibrinogen-like 2 POS NA

317 APAF1 apoptotic peptidase activating factor 1 POS NA

4671 NAIP NLR family, apoptosis inhibitory protein POS NA myeloid cell nuclear differentiation

4332 MNDA antigen POS NA

ATPase, H+ transporting, lysosomal V0

535 ATP6V0A1 subunit al POS NA

8790 FPGT fucose-1 -phosphate guanylyltransferase POS NA

4689 NCF4 neutrophil cytosolic factor 4, 40kDa POS NA colony stimulating factor 2 receptor, beta,

1439 CSF2RB low-affinity (granulocyte -macrophage) POS NA

965 CD58 CD58 molecule POS NA

25797 QPCT glutaminyl-peptide cyclotransferase POS NA dihydrolipoamide branched chain

1629 DBT transacylase E2 POS NA

1675 CFD complement factor D (adipsin) POS NA caspase 10, apoptosis-related cysteine

843 CASP10 peptidase POS NA glucosaminyl (N-acetyl) transferase 1 ,

2650 GCNT1 core 2 POS NA

11057 ABHD2 abhydrolase domain containing 2 POS NA

366 AQP9 aquaporin 9 POS NA

10201 NME6 non-metastatic cells 6, protein expressed POS NA in (nucleoside-diphosphate kinase)

1524 CX3CR1 chemokine (C-X3-C motif) receptor 1 POS NA

8875 VNN2 vanin 2 POS NA cAMP responsive element binding protein

9586 CREB5 5 POS NA

3101 HK3 hexokinase 3 (white cell) POS NA

9904 RBM19 RNA binding motif protein 19 POS NA

433 ASGR2 asialoglycoprotein receptor 2 POS NA

7409 VAV1 vav 1 guanine nucleotide exchange factor POS NA complement component (3b/4b) receptor 1

1378 CR1 (Knops blood group) POS NA

5724 PTAFR platelet-activating factor receptor POS NA

3034 HAL histidine ammonia-lyase POS NA

2745 GLRX glutaredoxin (thioltransferase) POS NA protein tyrosine phosphatase, non-receptor

5777 PTPN6 type 6 POS NA leukocyte immunoglobulin-like receptor,

subfamily A (without TM domain),

11026 LILRA3 member 3 POS NA

10326 SIRPB1 signal-regulatory protein beta 1 POS NA

1234 CCR5 chemokine (C-C motif) receptor 5 POS NA solute carrier family 25 (mitochondrial

8402 SLC25A11 carrier; oxoglutarate carrier), member 11 POS NA leukocyte immunoglobulin-like receptor,

subfamily B (with TM and ITIM

10859 LILRB1 domains), member 1 POS NA

27036 SIGLEC7 sialic acid binding Ig-like lectin 7 POS NA

2710 GK glycerol kinase POS NA tumor necrosis factor receptor

7132 TNFRSF1A superfamily, member 1A POS NA

4689 NCF4 neutrophil cytosolic factor 4, 40kDa POS NA

10288 LILRB2 leukocyte immunoglobulin-like receptor, POS NA subfamily B (with TM and ITIM

domains), member 2

ATPase, H+ transporting, lysosomal

537 ATP6AP1 accessory protein 1 POS NA leukocyte immunoglobulin-like receptor,

subfamily A (with TM domain), member

11024 LILRA1 1 POS NA

3055 HCK hemopoietic cell kinase POS NA

6916 TBXAS1 thromboxane A synthase 1 (platelet) POS NA

7916 BAT2 HLA-B associated transcript 2 POS NA

9074 CLDN6 claudin 6 POS NA leukocyte immunoglobulin-like receptor,

subfamily A (with TM domain), member

79168 LILRA6 6 POS NA

334 APLP2 amyloid beta (A4) precursor-like protein 2 POS NA

4218 RAB8A RAB8A, member RAS oncogene family POS NA

65220 NADK NAD kinase POS NA

65220 NADK NAD kinase POS NA

65220 NADK NAD kinase POS NA

23191 CYFIP1 cytoplasmic FMR1 interacting protein 1 POS NA

La ribonucleoprotein domain family,

23185 LARP4B member 4B POS NA

26234 FBXL5 F-box and leucine-rich repeat protein 5 POS NA

11151 COR01A coronin, actin binding protein, 1 A POS NA

2629 /// GBA /// glucosidase, beta, acid /// glucosidase,

2630 GBAP1 beta, acid pseudogene 1 POS NA

10548 TM9SF1 transmembrane 9 superfamily member 1 POS NA membrane bound O-acyltransferase

79143 MBOAT7 domain containing 7 POS NA

23208 SYT11 synaptotagmin XI POS NA

6809 STX3 syntaxin 3 POS NA

9470 EIF4E2 eukaryotic translation initiation factor 4E POS NA family member 2

ectonucleoside triphosphate

953 ENTPD1 diphosphohydrolase 1 POS NA

TNFSF12- TNFSF12-TNFSF13 readthrough ///

407977 /// TNFSF13 /// tumor necrosis factor (ligand)

8741 TNFSF13 superfamily, member 13 POS NA

5873 RAB27A RAB27A, member RAS oncogene family POS NA ash2 (absent, small, or homeotic)-like

9070 ASH2L (Drosophila) POS NA

3588 IL10RB interleukin 10 receptor, beta POS NA

11240 PADI2 peptidyl arginine deiminase, type II POS NA family with sequence similarity 65,

9750 FAM65B member B POS NA frequently rearranged in advanced T-cell

23401 FRAT2 lymphomas 2 POS NA

6404 SELPLG selectin P ligand POS NA

11314 CD300A CD300a molecule POS NA potassium large conductance calcium- activated channel, subfamily M, beta

3779 KCNMB1 member 1 POS NA

4688 NCF2 neutrophil cytosolic factor 2 POS NA caspase 1 , apoptosis-related cysteine

834 CASP1 peptidase (interleukin 1 , beta, convertase) POS NA lymphoblastic leukemia derived sequence

4066 LYL1 1 POS NA leukocyte immunoglobulin-like receptor,

subfamily B (with TM and ITIM

10288 LILRB2 domains), member 2 POS NA

7100 TLR5 toll-like receptor 5 POS NA integrin, alpha X (complement component

3687 ITGAX 3 receptor 4 subunit) POS NA

6252 RTN1 reticulon 1 POS NA 11078 /// NOLI 2 /// nucleolar protein 12 /// TRIO and F-actin

79159 TRIOBP binding protein POS NA tumor necrosis factor (ligand)

8741 TNFSF13 superfamily, member 13 POS NA solute carrier family 11 (proton-coupled

6556 SLC11A1 divalent metal ion transporters), member 1 POS NA

1488 CTBP2 C-terminal binding protein 2 POS NA

26118 WSB1 WD repeat and SOCS box-containing 1 POS NA

7841 MOGS mannosyl-oligosaccharide glucosidase POS NA

7940 LST1 leukocyte specific transcript 1 POS NA leukocyte-associated immunoglobulin-like

3903 LAIR1 receptor 1 POS NA leukocyte immunoglobulin-like receptor,

subfamily A (with TM domain), member

11024 LILRA1 1 POS NA

2358 FPR2 formyl peptide receptor 2 POS NA

2358 FPR2 formyl peptide receptor 2 POS NA leukocyte immunoglobulin-like receptor,

subfamily B (with TM and ITIM

11025 LILRB3 domains), member 3 POS NA tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein,

7531 YWHAE epsilon polypeptide POS NA leukocyte immunoglobulin-like receptor,

subfamily A (with TM domain), member

11027 LILRA2 2 POS NA leukocyte immunoglobulin-like receptor,

subfamily A (with TM domain), member

11027 LILRA2 2 POS NA leukocyte immunoglobulin-like receptor,

subfamily B (with TM and ITIM

11025 LILRB3 domains), member 3 POS NA 6452 SH3BP2 SH3 -domain binding protein 2 POS NA killer cell immunoglobulin-like receptor,

3803 KIR2DL2 two domains, long cytoplasmic tail, 2 POS NA serpin peptidase inhibitor, clade A (alpha-

5265 SERPINA1 1 antiproteinase, antitrypsin), member 1 POS NA

7940 LST1 leukocyte specific transcript 1 POS NA

5452 POU2F2 POU class 2 homeobox 2 POS NA

965 CD58 CD58 molecule POS NA potassium inwardly-rectifying channel,

3772 KCNJ15 subfamily J, member 15 POS NA

7879 RAB7A RAB7A, member RAS oncogene family POS NA

ATPase, H+ transporting, lysosomal

9114 ATP6V0D1 38kDa, V0 subunit dl POS NA interferon induced transmembrane protein

10410 IFITM3 3 (1-8U) POS NA

NIMA (never in mitosis gene a)- related

91754 NEK9 kinase 9 POS NA

2799 GNS glucosamine (N-acetyl)-6-sulfatase POS NA

ArfGAP with RhoGAP domain, ankyrin

116985 ARAP1 repeat and PH domain 1 POS NA

WD repeat and FYVE domain containing

23001 WDFY3 3 POS NA

23307 FKBP15 FK506 binding protein 15, 133kDa POS NA calmodulin regulated spectrin-associated

157922 CAMS API protein 1 POS NA ubiquitin protein ligase E3 component n-

23304 UBR2 recognin 2 POS NA transcription factor 7-like 2 (T-cell

6934 TCF7L2 specific, HMG-box) POS NA

10228 STX6 syntaxin 6 POS NA

6272 SORT1 sortilin 1 POS NA

23312 DMXL2 Dmx-like 2 POS NA 1955 MEGF9 multiple EGF-like-domains 9 POS NA

1955 MEGF9 multiple EGF-like-domains 9 POS NA

23151 GRAMD4 GRAM domain containing 4 POS NA

TAF4 RNA polymerase II, TATA box

binding protein (TBP)-associated factor,

6874 TAF4 135kDa POS NA

ARP3 actin-related protein 3 homolog

10096 ACTR3 (yeast) POS NA

ARP3 actin-related protein 3 homolog

10096 ACTR3 (yeast) POS NA

23328 SASH1 SAM and SH3 domain containing 1 POS NA

57205 ATP10D ATPase, class V, type 10D POS NA

10154 PLXNC1 plexin CI POS NA

3310 HSPA6 heat shock 70kDa protein 6 (HSP70B ^* ) POS NA

IQ motif containing GTPase activating

8826 IQGAP1 protein 1 POS NA

171546 C14orfl47 chromosome 14 open reading frame 147 POS NA

65220 NADK NAD kinase POS NA

9204 ZMYM6 zinc finger, MYM-type 6 POS NA

6398 SECTM1 secreted and transmembrane 1 POS NA

100289727 LOCI 00289727 hypothetical protein LOCI 00289727 ///

/// 653361 /// NCF1 /// neutrophil cytosolic factor 1 /// neutrophil

/// 654816 NCF1B /// cytosolic factor IB pseudogene ///

/// 654817 NCF1C neutrophil cytosolic factor 1C pseudogene POS NA

65220 NADK NAD kinase POS NA

339290 LOC339290 hypothetical LOC339290 POS NA osteosarcoma amplified 9, endoplasmic

10956 OS9 reticulum lectin POS NA

5606 MAP2K3 mitogen-activated protein kinase kinase 3 POS NA

7791 ZYX zyxin POS NA transcription factor 7-like 2 (T-cell

6934 TCF7L2 specific, HMG-box) POS NA transcription factor 7-like 2 (T-cell

6934 TCF7L2 specific, HMG-box) POS NA

1616 DAXX death-domain associated protein POS NA

2896 CRN granulin POS NA

1211 CLTA clathrin, light chain A POS NA

11196 SEC23IP SEC23 interacting protein POS NA calcium binding atopy-related autoantigen

10367 CBARA1 1 POS NA killer cell immunoglobulin-like receptor,

three domains, long cytoplasmic tail, 1 ///

killer cell immunoglobulin-like receptor,

three domains, long cytoplasmic tail, 2 ///

similar to killer cell immunoglobulin-like

receptor 3DL2 precursor (MHC class I

NK cell receptor) (Natural killer-

3811 /// KIR3DL1 /// associated transcript 4) (NKAT-4) (p70

3812 /// KIR3DL2 /// natural killer cell receptor clone CL-5)

727787 LOC727787 (CD 158k antigen) POS NA

23313 C22orf9 chromosome 22 open reading frame 9 POS NA

27036 SIGLEC7 sialic acid binding Ig-like lectin 7 POS NA

1890 TYMP thymidine phosphorylase POS NA

284021 C17orf60 chromosome 17 open reading frame 60 POS NA

Transcribed Transcribed

Locus Locus Transcribed Locus POS NA vacuolar protein sorting 35 homo log (S.

55737 VPS 35 cerevisiae) POS NA hematological and neurological expressed

51155 HN1 1 POS NA

10419 PRMT5 protein arginine methyltransferase 5 POS NA

UDP-N-acetyl-alpha-D- galactosamine:polypeptide N-

2590 GALNT2 acetylgalactosaminyltransferase 2 POS NA (GalNAc-T2)

51734 SEPX1 selenoprotein X, 1 POS NA

79180 EFHD2 EF-hand domain family, member D2 POS NA

51231 VR 3 vaccinia related kinase 3 POS NA

28958 CCDC56 coiled-coil domain containing 56 POS NA

54502 RBM47 RNA binding motif protein 47 POS NA

55577 NAGK N-acetylglucosamine kinase POS NA

80017 C14orfl59 chromosome 14 open reading frame 159 POS NA

114885 OSBPL11 oxysterol binding protein-like 11 POS NA

UDP-N-acetyl-alpha-D- galactosamine:polypeptide N- acetylgalactosaminyltransferase 7

51809 GALNT7 (GalNAc-T7) POS NA

51657 STYXL1 serine/threonine/tyrosine interacting-like 1 POS NA

VPS33B interacting protein, apical-

63894 VIPAR basolateral polarity regulator POS NA

SIL1 homo log, endoplasmic reticulum

64374 SIL1 chaperone (S. cerevisiae) POS NA

54840 APTX aprataxin POS NA

DCNl, defective in cullin neddylation 1,

54165 DCUN1D1 domain containing 1 (S. cerevisiae) POS NA

DNA-damage regulated autophagy

55332 DRAM1 modulator 1 POS NA

ATG7 autophagy related 7 homo log (S.

10533 ATG7 cerevisiae) POS NA

22921 MSRB2 methionine sulfoxide reductase B2 POS NA

80346 REEP4 receptor accessory protein 4 POS NA

80025 PANK2 pantothenate kinase 2 POS NA

80150 ASRGL1 asparaginase like 1 POS NA tubulin polymerization-promoting protein

51673 TPPP3 family member 3 POS NA

64342 HS1BP3 HCLS 1 binding protein 3 POS NA cat eye syndrome chromosome region,

51816 CECR1 candidate 1 POS NA

ATP -binding cassette, sub-family A

10347 ABCA7 (ABC1), member 7 POS NA paired immunoglobin-like type 2 receptor

29992 PILRA alpha POS NA

56935 Cl lorf75 chromosome 11 open reading frame 75 POS NA frequently rearranged in advanced T-cell

10023 FRAT1 lymphomas POS NA

80167 C4orf29 chromosome 4 open reading frame 29 POS NA

8778 SIGLEC5 sialic acid binding Ig-like lectin 5 POS NA purinergic receptor P2Y, G-protein

53829 P2RY13 coupled, 13 POS NA

55911 APOB48R apolipoprotein B48 receptor POS NA nucleotide-binding oligomerization

64127 NOD2 domain containing 2 POS NA

55711 FAR2 fatty acyl CoA reductase 2 POS NA

79670 ZCCHC6 zinc finger, CCHC domain containing 6 POS NA

78996 C7orf49 chromosome 7 open reading frame 49 POS NA

81876 RAB1B RAB1B, member RAS oncogene family POS NA

406991 /// MIR21 /// microRNA 21 /// transmembrane protein

81671 TMEM49 49 POS NA

81609 SNX27 sorting nexin family member 27 POS NA anterior pharynx defective 1 homolog B

83464 APH1B (C. elegans) POS NA

9236 CCPG1 cell cycle progression 1 POS NA

58528 RRAGD Ras-related GTP binding D POS NA linker for activation of T cells family,

7462 LAT2 member 2 POS NA

29100 TMEM208 transmembrane protein 208 POS NA

29108 PYCARD PYD and CARD domain containing POS NA

93129 ORAI3 ORAI calcium release-activated calcium POS NA modulator 3

7702 ZNF143 zinc finger protein 143 POS NA

158747 MOSPD2 motile sperm domain containing 2 POS NA

283232 TMEM80 transmembrane protein 80 POS NA

9236 CCPG1 cell cycle progression 1 POS NA solute carrier family 27 (fatty acid

11000 SLC27A3 transporter), member 3 POS NA paired immunoglobin-like type 2 receptor

29992 PILRA alpha POS NA vacuolar protein sorting 35 homo log (S.

55737 VPS 35 cerevisiae) POS NA

79180 EFHD2 EF-hand domain family, member D2 POS NA

54502 RBM47 R A binding motif protein 47 POS NA

26253 CLEC4E C-type lectin domain family 4, member E POS NA family with sequence similarity 45,

404636 /// FAM45 A /// member A /// family with sequence

55855 FAM45B similarity 45, member A pseudogene POS NA

10890 RAB10 RAB10, member RAS oncogene family POS NA

10890 RAB10 RAB10, member RAS oncogene family POS NA

54926 UBE2R2 ubiquitin-conjugating enzyme E2R 2 POS NA

27166 PRELID1 PRELI domain containing 1 POS NA

84522 JAGN1 jagunal homo log 1 (Drosophila) POS NA

COX15 homolog, cytochrome c oxidase

1355 C0X15 assembly protein (yeast) POS NA

25798 BRI3 brain protein 13 POS NA

N-acetylneuraminate pyruvate lyase

80896 NPL (dihydrodipicolinate synthase) POS NA

51192 CKLF chemokine-like factor POS NA

84282 R F135 ring finger protein 135 POS NA family with sequence similarity 126,

84668 FAM126A member A POS NA

9424 KCNK6 potassium channel, subfamily K, member POS NA 6

91662 NLRP12 NLR family, pyrin domain containing 12 POS NA dehydrogenase/reductase (SDR family)

10170 DHRS9 member 9 POS NA

54675 CRLS1 cardiolipin synthase 1 POS NA dehydrogenase/reductase (SDR family)

10170 DHRS9 member 9 POS NA

ATG7 autophagy related 7 homo log (S.

10533 ATG7 cerevisiae) POS NA transmembrane protein with EGF-like and

23671 TMEFF2 two follistatin-like domains 2 POS NA

84108 PCGF6 polycomb group ring finger 6 POS NA membrane-spanning 4-domains,

58475 MS4A7 subfamily A, member 7 POS NA

7077 TIMP2 TIMP metallopeptidase inhibitor 2 POS NA

90231 KIAA2013 KIAA2013 POS NA

10238 DCAF7 DDB1 and CUL4 associated factor 7 POS NA male-specific lethal 1 homolog

339287 MSL1 (Drosophila) POS NA

92181 UBTD2 ubiquitin domain containing 2 POS NA polymerase (RNA) I polypeptide D,

51082 POLR1D 16kDa POS NA

170954 KIAA1949 KIAA1949 POS NA

57085 AGTRAP angiotensin II receptor-associated protein POS NA serine palmitoyltransferase, long chain

9517 SPTLC2 base subunit 2 POS NA

8540 AGPS alkylglycerone phosphate synthase POS NA

55795 PCID2 PCI domain containing 2 POS NA

414 ARSD arylsulfatase D POS NA

284996 RNF149 ring finger protein 149 POS NA

MOBl, Mps One Binder kinase activator¬

126308 MOBKL2A like 2A (yeast) POS NA 286144 C8orf83 chromosome 8 open reading frame 83 POS NA

8935 SKAP2 src kinase associated phosphoprotein 2 POS NA interferon (alpha, beta and omega)

3454 IFNAR1 receptor 1 POS NA

100133150 FLJ45340 ///

/// FLJ45445 /// hypothetical LOC402483 /// hypothetical

100287274 LOC100133150 LOC399844 /// hypothetical

/// 399844 /// LOC 100133150 /// hypothetical

/// 402483 LOCI 00287274 LOCI 00287274 /// hypothetical

/// 653340 /// LOC653340 LOC653340 POS NA

203228 C9orf72 chromosome 9 open reading frame 72 POS NA

79689 STEAP4 STEAP family member 4 POS NA murine retrovirus integration site 1

10335 MRVI1 homolog POS NA

6850 SYK spleen tyrosine kinase POS NA

219854 TMEM218 transmembrane protein 218 POS NA

DNA fragmentation factor, 45kDa, alpha

1676 DFFA polypeptide POS NA

57688 ZSWIM6 zinc finger, SWIM-type containing 6 POS NA

64420 SUSD1 sushi domain containing 1 POS NA phosphoinositide-3 -kinase adaptor protein

118788 PIK3AP1 1 POS NA p21 protein (Cdc42/Rac)-activated kinase

5058 PAK1 1 POS NA

121274 ZNF641 zinc finger protein 641 POS NA

57187 THOC2 THO complex 2 POS NA

221955 DAGLB diacylglycerol lipase, beta POS NA

91612 CHURC1 churchill domain containing 1 POS NA solute carrier family 12

(potassium/chloride transporters), member

9990 SLC12A6 6 POS NA

219972 MPEG1 Macrophage expressed 1 POS NA 219972 MPEG1 macrophage expressed 1 POS NA

157697 ERICH 1 glutamate-rich 1 POS NA

150290 DUSP18 dual specificity phosphatase 18 POS NA

402483 FLJ45340 hypothetical LOC402483 POS NA

27163 NAAA N-acylethanolamine acid amidase POS NA

5724 PTAFR platelet-activating factor receptor POS NA membrane bound O-acyltransferase

154141 MBOAT1 domain containing 1 POS NA protein tyrosine phosphatase, receptor

5795 PTPRJ type, J POS NA eukaryotic elongation factor,

60678 EEFSEC selenocysteine-tRNA-specific POS NA

54790 TET2 tet oncogene family member 2 POS NA potassium voltage-gated channel, Isk-

10008 KCNE3 related family, member 3 POS NA

Eukaryotic translation initiation factor 2C,

192670 EIF2C4 4 POS NA

374395 TMEM179B transmembrane protein 179B POS NA adhesion molecule, interacts with

120425 AMICA1 CXADR antigen 1 POS NA protein phosphatase, Mg2+/Mn2+

151742 PPM1L dependent, 1L POS NA signal transducing adaptor molecule (SH3

10254 STAM2 domain and IT AM motif) 2 POS NA

29097 CNIH4 cornichon homolog 4 (Drosophila) POS NA ubiquitin associated and SH3 domain

84959 UBASH3B containing B POS NA

11057 ABHD2 abhydrolase domain containing 2 POS NA

55356 SLC22A15 solute carrier family 22, member 15 POS NA

128346 Clorfl62 chromosome 1 open reading frame 162 POS NA sarcoglycan, delta (35kDa dystrophin-

6444 SGCD associated glycoprotein) POS NA serpin peptidase inhibitor, clade B

1992 SERPINB1 (ovalbumin), member 1 POS NA solute carrier family 6 (neurotransmitter

6533 SLC6A6 transporter, taurine), member 6 POS NA

CD36 molecule (thrombospondin

948 CD36 receptor) POS NA

150166 LOC150166 hypothetical protein LOCI 50166 POS NA

120892 LRR 2 leucine-rich repeat kinase 2 POS NA

144423 GLT1D1 glycosyltransferase 1 domain containing 1 POS NA

401115 C4orf48 chromosome 4 open reading frame 48 POS NA

ST8 alpha-N-acetyl-neuraminide alpha-

7903 ST8SIA4 2,8-sialyltransferase 4 POS NA

FYVE, RhoGEF and PH domain

121512 FGD4 containing 4 POS NA glucosaminyl (N-acetyl) transferase 2, 1-

2651 GCNT2 branching enzyme (I blood group) POS NA

ST8 alpha-N-acetyl-neuraminide alpha-

7903 ST8SIA4 2,8-sialyltransferase 4 POS NA amyloid beta (A4) precursor protein- binding, family B, member 1 interacting

54518 APBB1IP protein POS NA

729082 LOC729082 hypothetical protein LOC729082 POS NA

64333 ARHGAP9 Rho GTPase activating protein 9 POS NA

CAMP responsive element binding

9586 CREB5 protein 5 POS NA

153684 LOC153684 hypothetical LOCI 53684 POS NA

GIY-YIG domain containing 1 /// GIY- YIG domain containing 2 ///

445329 /// GIYD1 /// sulfotransferase family, cytosolic, 1A,

548593 /// GIYD2 /// phenol-preferring, member 3 ///

6818 /// SULT1A3 /// sulfotransferase family, cytosolic, 1A,

79008 SULT1A4 phenol-preferring, member 4 POS NA 53916 RAB4B RAB4B, member RAS oncogene family POS NA

10169 /// C15orf63 /// chromosome 15 open reading frame 63 ///

25764 SERF2 small EDRK-rich factor 2 POS NA

51657 STYXL1 serine/threonine/tyrosine interacting-like 1 POS NA

7227 TRPS1 trichorhinophalangeal syndrome I POS NA nudix (nucleoside diphosphate linked

131870 NUDT16 moiety X)-type motif 16 POS NA osteopetrosis associated transmembrane

28962 OSTM1 protein 1 POS NA solute carrier family 8 (sodium/calcium

6546 SLC8A1 exchanger), member 1 POS NA

Proline rich Gla (G-carboxyglutamic acid)

79056 PR G4 4 (transmembrane) POS NA solute carrier family 8 (sodium/calcium

6546 SLC8A1 exchanger), member 1 POS NA

8562 DENR density-regulated protein POS NA calcineurin-like phosphoesterase domain

55313 CPPED1 containing 1 POS NA

5947 RBP1 Retinol binding protein 1 , cellular POS NA

84314 TMEM107 transmembrane protein 107 POS NA

57654 KIAA1530 KIAA1530 POS NA

84106 PRAM1 PML-RARA regulated adaptor molecule 1 POS NA

ST8 alpha-N-acetyl-neuraminide alpha-

7903 ST8SIA4 2,8-sialyltransferase 4 POS NA

DEAD (Asp-Glu-Ala-Asp) box

23586 DDX58 polypeptide 58 POS NA capping protein (actin filament) muscle Z-

832 CAPZB line, beta POS NA

10636 RGS14 regulator of G-protein signaling 14 POS NA

7942 TFEB transcription factor EB POS NA

57175 COROIB coronin, actin binding protein, IB POS NA

158747 MOSPD2 motile sperm domain containing 2 POS NA 89790 /// SIGLEC10 /// sialic acid binding Ig-like lectin 10 /// 89858 SIGLEC12 sialic acid binding Ig-like lectin 12 POS POS

10935 PRDX3 peroxiredoxin 3 POS POS

ATPase, H+ transporting, lysosomal

523 ATP6V1A 70kDa, VI subunit A POS POS

22918 CD93 CD93 molecule POS POS

1436 CSF1R colony stimulating factor 1 receptor POS POS

1612 DAPK1 death-associated protein kinase 1 POS POS

6416 MAP2K4 mitogen-activated protein kinase kinase 4 POS POS

NADH dehydrogenase (ubiquinone) 1

4709 NDUFB3 beta subcomplex, 3, 12kDa POS POS

Bruton agammaglobulinemia tyrosine

695 BTK kinase POS POS potassium inwardly-rectifying channel,

3759 KCNJ2 subfamily J, member 2 POS POS

6282 S100A11 SI 00 calcium binding protein Al 1 POS POS

5052 PRDX1 peroxiredoxin 1 POS POS

5175 PEC AMI platelet/endothelial cell adhesion molecule POS POS proline synthetase co-transcribed homolog

11212 PROSC (bacterial) POS POS proline synthetase co-transcribed homolog

11212 PROSC (bacterial) POS POS methylcrotonoyl-CoA carboxylase 2

64087 MCCC2 (beta) POS POS

124565 SLC38A10 solute carrier family 38, member 10 POS POS

79789 CLMN calmin (calponin-like, transmembrane) POS POS

10066 SCAMP2 secretory carrier membrane protein 2 POS POS

548593 /// GIYD1 /// GIY-YIG domain containing 1 /// GIY- 79008 GIYD2 YIG domain containing 2 POS POS

29095 ORMDL2 ORMl-like 2 (S. cerevisiae) POS POS

ArfGAP with RhoGAP domain, ankyrin

64411 ARAP3 repeat and PH domain 3 POS POS phosphopantothenoylcysteine

60490 PPCDC decarboxylase POS POS sema domain, immunoglobulin domain

(Ig), transmembrane domain (TM) and

short cytoplasmic domain, (semaphorin)

64218 SEMA4A 4A POS POS

51192 CKLF chemokine-like factor POS POS

27141 CIDEB cell death-inducing DFFA-like effector b POS POS

91860 CALML4 calmodulin-like 4 POS POS

51231 VR 3 vaccinia related kinase 3 POS POS diacylglycerol O-acyltransferase homolog

84649 DGAT2 2 (mouse) POS POS

113402 SFT2D1 SFT2 domain containing 1 POS POS

113402 SFT2D1 SFT2 domain containing 1 POS POS

FYVE, RhoGEF and PH domain

121512 FGD4 containing 4 POS POS

64778 FNDC3B fibronectin type III domain containing 3B POS POS

55072 R F31 Ring finger protein 31 POS POS

57823 SLAMF7 SLAM family member 7 POS POS

158402 LOC158402 hypothetical protein LOCI 58402 POS POS complement component (3b/4b) receptor 1

1378 CR1 (Knops blood group) POS POS

Ingenuity pathway analysis of the genes that were either positively or negatively correlated with HAI titers showed enrichment for genes related to the cell-mediated immune response and to the infection mechanism and inflammatory response, respectively (Fig. 7). The identification of genes such as TLR5, CASPl, PYCARD, NOD2 and NAIP suggested previously unknown mechanistic links between host innate immunity and humoral responses to influenza vaccination. A candidate vaccine against influenza composed of a recombinant fusion protein linking influenza antigens to the Toll-like receptor 5 ligand fiagellin may induce potent immunogenicity in mice and humans. In addition, canonical pathways, such as T cell receptor antigen receptor signaling and CTLA-4 signaling in cytotoxic T lymphocytes, included many of the genes present in the cell-mediated immune response network and were among those with the highest enrichment score by ingenuity pathway analysis. This data indicates a possible association between cellular responses and humoral responses to vaccination with TIV. Among the top canonical pathways enriched for genes positively correlated to HAI response, networks associated with innate immunity were found, such as the natural killer cell signaling network, and network for the production of nitric oxide and reactive oxygen species in macrophages (Fig 8A, B). Analysis also showed that the expression of interferon-related genes (including those encoding the receptors for interferon-a and interferon-γ) on day 3 after vaccination was correlated to the HAI response (Fig. 9), which suggested a link between the interferon response and the antibody response.

The genes whose expression correlated with the HAI response at day 28 after vaccination of subjects were compared with TIV with the genomic signatures of the cells of the immune response defined by our meta-analysis. This approach showed that the set of genes positively correlated to HAI response was enriched for genes with high expression in B cells (Fig. 4C) and, more specifically, in the ASC subset (Fig. 4D). The genes with negative correlation to the HAI response were substantially enriched among the genes with high expression in T cells (Fig. 4C), which supported the identification of the T cell pathways by ingenuity pathway analysis (Fig. 8A, B). Together these data demonstrated the identification of early signatures that correlated with later HAI titers induced by TIV.

Molecular signatures to predict antibody responses

Once signatures that correlated with the magnitude of HAI response were delineated our next step was to identify the minimum sets of genes one could use to predict such a response. Ideally, such sets of genes must be able to be used to accurately classify high responders versus low responders in additional and independent TIV trials. For this, we used DAMIP (discriminant analysis via mixed integer programming), which is a very powerful supervised-learning classification method for predicting various biomedical and 'biobehavioral' phenomena.

In initial analyses, the subjects vaccinated with TIV into were classified two 'extreme' groups: very low HAI responders, and very high HAI responders. The former group consisted of subjects with an increase of twofold or less in HAI titers against any of the three influenza strains of the vaccine (Fig. 1 A). The latter group consisted of subjects with an increase of eightfold or more in the HAI response for at least one of the three influenza strains of the vaccine. Subjects with intermediate HAI response (between twofold and eightfold) and subjects for whom microarray data were not available at either day 3 or day 7 after vaccination (n = 7) were not analyzed. The 2008-2009 trial was used that to train the DAMIP model to establish an unbiased estimate of correct classification. A second, independent trial, was used to evaluate the predictive accuracy of the classification rules identified in the first trial (Fig. 5a). The second trial (the 2007-2008 trial) consisted of the microarray gene-expression profiles of subjects (n = 9) vaccinated with TIV in the previous year. With this approach, DAMIP model identified 12 sets of genes containing two to four genes each (each set associates with one predictive rule) from

2008- 2009 trial with a tenfold cross-validation accuracy over 90%. The resulting 'blind prediction' accuracy of the 2007-2008 trial (predicting low or high responders) was over 90%. Furthermore, some of the 271 sets of discriminatory genes offered an accuracy of over 90% in both tenfold cross-validation in the training trial and 'blind prediction' accuracy (Fig. 5 A).

Real-time RT-PCR was used to confirm that 44 genes from the DAMIP gene signatures encoded molecules with potential biological relevance and/or utility as a predictor of influenza vaccine immunogenicity. A significant positive correlation (r = 0.679; P = 3.25 x 10-12) was found for changes in expression on day 3 or 7 relative to baseline expression as detected by microarray and RT-PCR (Fig. 5B), which confirmed the correctness of the microarray data. More notably, that result gave confidence to test some of the candidate predictors of immunogenicity in a third and independent influenza vaccine trial (Fig. 5A). We collected RNA from PBMCs of subjects (n = 30) vaccinated with TIV during the 2009-2010 influenza season and analyzed this RNA by real-time RT- PCR. We then used the expression of the 44 genes selected from the initial DAMIP gene signatures to confirm their utility in predicting the magnitude of antibody response in this third TIV trial (Fig. 5 A). To avoid the identification of 'over-trained' rules, the DAMIP analysis were re-ran using the 2008-2009 trial as the training set and the 2007-2008 and

2009- 2010 trials as the blind predictive sets. This approach identified 47 sets of genes; some of these were used to correctly classify >85%> of the vaccines as being very low HAI responders or very high HAI responders in any of the three trials.

Because seroconversion after vaccination is widely defined as a fourfold increase in HAI titers, an additional DAMIP analysis was ran using a cutoff of fourfold to classify the vaccinees (Fig. 5A). Thus, subjects were classified with an increase of fourfold or greater in the HAI titers after vaccination as 'high responders' and those with an increase of twofold or less as 'low responders'. With the 2008-2009 trial as a training set and 2007- 2008 and 2009-2010 trials as blind predictive sets, the DAMIP model generated 42 sets of gene signatures (Fig. 5C), each composed of three to four discriminatory genes, some of which had an unbiased estimate of correct classification above 85%, as determined by tenfold cross-validation and blind prediction.

Table 2. Genes that appeared in the third analysis of the DAMIP model

leukocyte immunoglobulin-like receptor, subfamily

LILRA6 A (with TM domain), member 6 Day 3 2 leukocyte immunoglobulin-like receptor, subfamily

LILRB2 B (with TM and ITIM domains), member 2 Day 3 2

ANPEP alanyl (membrane) aminopeptidase Day 3 1 leukocyte immunoglobulin-like receptor, subfamily

LILRB1 B (with TM and ITIM domains), member 1 Day 3 1

HSP90B1 heat shock protein 90kDa beta (Grp94),

HSP90B1 member 1 Day 7 1 immunoglobulin heavy constant gamma 1 (Glm

IGHG1 marker) Day 7 1

PDIA6 protein disulfide isomerase family A, member 6 Day 7 1

HYOU1 hypoxia up-regulated 1 Day 7 1

Among the genes in the TIV DAMIP models, five are members of the leukocyte immunoglobulin-like receptor family. These genes are expressed by immune-response cells of both myeloid and lymphoid lineages and the molecules they encode are thought to have an immunomodulatory role in the innate and adaptive immune systems by regulating T cells and autoimmunity. Meta-analysis showed that these genes had high expression in monocytes and myeloid DCs at day 3 after vaccination. These results and the presence of five members of this family among markers of antibody responses to influenza vaccination raised the possibility of previously unknown roles for these innate immune receptors in regulating antibody responses .

CaMKIV regulates the antibody response

To demonstrate that the gene signatures identified could be used to generate new hypotheses, CAMK4 was selected for functional confirmation experiments. CaMKIV is involved in several processes of the immune system, such as T cell development, inflammatory responses, and the maintenance of hematopoietic stem cells. However, nothing is known about the possible role of CaMKIV in B cell responses.

The change in CAMK4 expression on day 3 after vaccination with TIV was negatively correlated with the antibody response on day 28 after vaccination in two independent trials (Fig. 6A). Additionally, the change in CAMK4 expression was negatively correlated with the population expansion of IgG-secreting plasmablasts at day 7 (Fig. 6B), which suggested a possible role for CaMKIV in the regulation of antibody responses to vaccination against influenza.

In vitro stimulation of mouse splenocytes with TIV resulted in phosphorylation of CaMKIV (Fig. 6C), which suggested that this vaccine may trigger activation of CaMKIV. That finding was further demonstrated in human PBMCs, in which in vitro stimulation with influenza vaccine resulted in phosphorylation of CaMKIV as early as 2 h after stimulation (Fig. 6D). The mechanism by which this occurs remains to be identified.

To check if CaMKIV regulates the antibody response to influenza vaccine, wild- type and Camk4-/- mice were immunized with TIV and measured serum concentrations of IgGl and IgG2c on days 7, 14 and 28 after vaccination (Fig. 6E). After immunization, Camk4-/- mice had a significantly greater antibody response than that of wild-type mice (Fig. 6E). The biggest difference was on day 7, with 3- to 6.5-fold higher antibody titers in Camk4-/- mice than in wild-type mice (Fig. 6E). These results supported our prediction based on the microarray results and suggested that CaMKIV is important in the regulation of B cell response.

Clinical study.

This study included subjects vaccinated with TIV during the three consecutive influenza seasons in 2008-2009 (trial \; n = 28), 2007-2008 (trial 2; n = 9) and 2009-

2010 (trial 3; n = 30); one trial included subjects vaccinated with LAIV during 2008-2009 influenza season. Young healthy adults 18-50 years old were vaccinated with one dose of TIV (Fluarix (Glaxo SmithKline Biologicals) in 2007-2008 and 2008-2009 or Fluvirin (Novartis Vaccines and Diagnostics) in 2009-2010) or LAIV (FluMist; Medlmmune) according to guidelines for influenza vaccination (influenza virus strains in the vaccines). Written informed consent was obtained from each subject with institutional review and approval from the Emory University Institutional Review Board.

Cells, plasma and RNA isolation.

PBMCs and plasma were isolated from fresh blood (in Vacutainer cell preparation tubes with sodium citrate) according to the manufacturer's protocol (BD). Total RNA from fresh PBMCs (-1.5 10 ⁶ cells) was purified with TRIzol according to the manufacturer's instructions (Invitrogen, Life Technologies). The purity of all RNA samples was checked with an ND-1000 spectrophotometer (NanoDrop Technologies) and their integrity was checked by electrophoresis on a 2100 BioAnalyzer (Agilent Technologies).

RT-PCR.

Total R A was reverse-transcribed with a High-Capacity cDNA Archive Kit

Protocol (Applied Biosystems). Custom-designed Low Density Array and Quantitative Real-Time PCR Analysis Low Density Arrays for 48 genes were from Applied

Biosystems. Microarray experiments.

Total RNA was hybridized on Human U133 Plus 2.0 arrays (with a GeneTitan platform, Affymetrix or individual cartridges). The intensity microarray data for probe sets (called 'genes' here) was normalized by the robust multi-array average method, which includes global background adjustment and quantile normalization. For microarray analysis of flow cytometry-sorted subsets of immune-response cells, total RNA was amplified with the WT-Ovation Pico RNA Amplification System according to the manufacturer's instructions (NuGEN). Next, cDNA was labeled with an FL-Ovation cDNA Biotin Module V2 kit according to the manufacturer's protocol (NuGEN). The Affymetrix GeneChip Array Station automation platform was used for hybridization to Affymetrix HT Human Genome U133A arrays (Affymetrix) and washing. Arrays were scanned on a GeneChip HT Array Plate Scanner (00-0332; Affymetrix) and data were processed with normalization by robust multi-array averaging.

HAI assays.

Plasma samples were treated with receptor-destroying enzyme (Denka Seiken) at a ratio of one part plasma to three parts enzyme and incubation overnight at 37 °C. The next morning, the enzyme was inactivated by incubation of the samples for 1 h at 56 °C.

Samples were then serially diluted with PBS in 96-well V-bottomed plates (Nunc), and 4 hemagglutinating units of influenza strain HlNl or H3N2 or influenza B virus was added to each well. After 30 min at 25 °C, 50 μΐ of 0.5% turkey RBCs (Rockland

Immunochemicals) suspended in PBS with 0.5% (wt/vol) BSA was added to each well and plates were shaken manually. After an additional 30 min at 25 °C, plasma titers were measured as the reciprocal of the final dilution for which a pellet was observed. Negative and positive control plasmas for each virus were used for reference. B cell ELISPOT and flow cytometry of B cells.

First, 96-well ELISPOT filter plates (MAHA N4510; Millipore) were coated overnight with vaccine against influenza (same vaccine as that used for donor vaccination) at a dilution of 1 :20 in PBS or with goat antibody to human immunoglobulin (HI 7000; Caltag). Plates were washed, and unoccupied sites on the filters were blocked by incubation for 2 h at 37 °C with complete RPMI medium containing 10% (vol/vol) FCS. Purified and extensively washed PBMCs were added to the plates in serial dilutions, followed by incubation for 6 h or overnight. Plates were washed with PBS followed by PBS containing 0.05% (vol/vol) Tween and then were incubated for 1.5 h at 25 °C with biotinylated antibody to human IgG (HI 0015; Invitrogen), IgA (H14015; Invitrogen) or IgM (H15015; Invitrogen). After being washed, plates were incubated with an avidin D- horseradish peroxidase conjugate (A-2004; Vector Laboratories) and were finally developed with AEC substrate (3 amino-9 ethylcarbozole; Sigma). Developed plates were scanned and analyzed with an automated ELISPOT counter (Cellular Technologies). Flow cytometry analysis was done on whole blood. Before analysis, red blood cells were lysed twice (4 min each) at 25 °C by incubation with BD FACS Lysing Solution (Becton Dickinson). Blood (300-400 μΐ) was then incubated for 30 min at 25 °C with the appropriate antibodies: antibody to CD3 (anti-CD3; 557851; Pharmingen), anti-CD20 (335793; Pharmingen), anti-CD38 (555460; Pharmingen), anti-CD19 (555412;

Pharmingen) and anti-CD27 (17-0279; eBioscience). ASCs were gated and isolated as CD19 ⁺ CD3 CD20 ^{lD neg} CD27 ^hi CD38 ^hi cells. Flow cytometry data were analyzed with Flow Jo software. Analysis of immune responses in Camk4 ^~ ' ^~ mice.

C57BL/6 mice 8-12 weeks old were from The Jackson Laboratory. Camk4 ¹ mice were generated and were back-bred to C57BL/6J mice for more than 12 generations. Mice were immunized in the right and left hamstring muscles with the human Fluvirin vaccine (Novartis Vaccines and Diagnostics) diluted 1 :5. Blood was obtained from mice at various time points for analysis of influenza-specific antibody responses. All animal procedures were done in accordance with guidelines established by the Institutional Animal Care and Use Committee of Duke University.

For enzyme-linked immunosorbent assay, 96-well Maxisorp plates (Nunc) were coated overnight at 4 °C with Fluvirin (for trials in 2009-2010 and 2010-2011; 2 μg/ml in a volume of 100 μΐ; Novartis Vaccines and Diagnostics). Plates were washed three times with 5% (vol/vol) Tween 20 in PBS with a Biotek Auto Plate Washer, followed by incubation for 2 h at 25 °C with 200 μΐ per well of a solution of 4% (wt/vol) nonfat dry milk (Bio-Rad). Serum samples obtained from immunized mice at various time points were diluted (1 : 100) in 0.1% (wt/vol) nonfat dry milk in a solution of 0.5% (vol/vol) Tween 20 in PBS and were incubated for 2 h at 25 °C on blocked plates. Plates were washed five times after incubation of sample serum, and horseradish peroxidase- conjugated antibody to mouse IgG2c (1 :2,000 dilution; 1079-05; SouthemBiotech) and to mouse IgGl (1 :5,000 dilution; 1070-05; SouthemBiotech) in 0.5% (vol/vol) Tween-20 in PBS were added, then plates were incubated for 2 h at 25 °C. Plates were washed seven times with 0.5%> (vol/vol) Tween-20 in PBS and were developed with

tetramethylbenzidine substrate (100 μΐ per well; BD Biosciences), then reactions were stopped with 2 N H ₂ SO ₄ . Plates were analyzed with a BioRad plate reading

spectrophotometer at 450 nm with correction at 595 nm. Results are presented as absorbance at 450 nm.