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Fig. 7. 2-fold changed genes involved in theImmune response. Red are upregulated (ORF2 vs. vector); Green are downregulatedgenes; the intensity of color indicates the degree of down or upregulation; cytokine; enzyme; transcription regulatior; transporter.

Fig. 6. Genes with 2-fold altered expression involved in the gastrointestinal diseases.Red are upregulated (ORF2 vs. vector); Green are downregulated genes; the intensity of color indicates the degree of down or upregulation; cytokine; enzyme; transcription regulatior; transporter.

Fig. 5. Genes with 2-fold altered expression participated in the pathway of leukocyteextravasation signaling. Genes in red areupregulated (ORF2 vs. vector); and the greenare downregulated genes; enzyme; group or complex; kinase; other.

Fig. 4. Genes with 2-fold altered expression participated in the antigen presentation pathway. Genes in red are upregulated (ORF2 vs. vector); green are downregulated genes; represents transmembrane receptor; represents transcription regulation.

Genome-wide Analysis of Gene Expression Profiles Induced by Norwalk Virus Capsid Protein Gene

P. Liu1. YH. Li2, H. Hsiao1, N. Wagar2, C. L Moe1

1Rollins School of Public Health, 2Yerkes National Primate Research Center, Emory University, Atlanta, GA 30322

Noroviruses (NoV) are the leading cause of outbreaks of non-bacterial gastroenteritis both in the US and worldwide. Currently, a vaccine is not available, but several vaccine strategies target the norovirus capsid protein encoded by the open reading frame 2 (ORF-2). Recombinant norovirus capsid protein can self-assemble into virus-like particles (VLP) that are antigenically and morphologically similar to native whole norovirus. In order to better understand the molecular mechanism of ORF-2 as a norovirus vaccine candidate, we transfected Norwalk virus ORF-2 plasmid DNA into a human intestinal cell line Caco-2. Analysis by SYBR green real-time PCR showed that ORF2 expression increased 5,039~27,364-fold in the ORF2-transfected cells compared to cells transfected with only the vector. The whole genome expression profiles were analyzed in triplicate experiments using an Affymetrix human U133_ 2.0 array representing approximately 47,000 transcripts. A total of 3,483 transcripts exhibited at least 1.5-fold differences in their expression levels. Statistical analyses indicated that 54 transcripts were significantly different (p<0.05) between ORF2-transfected and vector only-transfected cells. Out of these, 28 mRNAs were up-regulated while 26 mRNAs were down-regulated. Hierarchical clustering indicated that the gene expression patterns of ORF2- and vector-transfected cells could be clearly distinguished. We found that zinc finger protein 236 (ZNF236), zinc finger protein 519 (ZNF519) and B-cell CLL/lymphoma 10 (BCL10) were the most important up-regulated genes in this study. Signal pathway analysis showed that the zinc finger proteins are involved in transcriptional regulation and that BCL10 plays a key role in antigen-receptor-mediated lymphocyte activation and the development of B and T cells. ORF2 transfection resulted in the down-regulation of chemokine receptor 1 (CCR1) and tumor necrosis factor superfamily member 8 (TNFSM8), both genes involved in innate and adaptive immune responses. These results provide novel information on the complex immune response at the transcriptional level in human intestinal cells induced by a norovirus vaccine candidate antigen.

Fig. 2. Hierarchical clustering of 134 geneswith statistical significant different expressionbetween ORF2 transfected cells and vector only transfected cells. Each row represents a single gene; each column represents either ORF2 transfected or vector transfected sample;red color is up-regulated genes and green is down-regulated genes in ORF2 transfected versus vector transfected cells

Fig. 1. ORF2 expression in Caco-2mammalian cells with triplicate experiments.SYBR green real-time PCR showed thatORF2 expression increased 104-105.8-fold in the ORF2-transfected Caco-2 cells compared to the vector alone transfected cells.

ORF2 CloneWe obtained a gift NV ORF2 clone that ORF2 fragment was cloned into apSPORT expression vector, and also a blank vector from Dr. Kim Green at NIH. Both ORF2 and pSPORT plasmid DNAs were transformed to E.coli and the DNAwas confirmed by endonuclease digestion. Caco-2 cell culture and DNA transfectionA human intestinal cell monolayer (Caco-2) was used as our in vitro mode oftransfection with the NV ORF2 expression vector and pSPORT vector. Caco-2cells (ATCC, VA) was grown in Dulbecco's Modified Eagle's Mediumsupplemented with 1 % nonessential amino acids, 10 % fetal calf serum (FCS),1000 U/liter penicillin, and 1 mg/liter streptomycin. NV ORF2 and pSPORT vectorDNA were transfected to Caco-2 cells using lipofectamine 2000 reagent on threeseparate days. RNA was extracted from the transfected cells Total TNA extraction and cRNA synthesisTotal RNAs from Caco-2 cells were prepared with the RNeasy Mini kit (Qiagen Inc,CA). and their integrities were verified on an Agilent Bioanalyzer. First-strandcDNAs were synthesized from 5 µg of each RNA sample. After RNase H-mediatedsecond-strand cDNA synthesis, double-stranded cDNAs were purified andbiotinylated complementary RNAs (cRNAs) were generated by an in vitrotranscription. Biotinylated cRNAs were cleaned up, fragmented, and hybridized toAffymetrix Human Genome U133 Plus 2.0 Array chips. Chips were processedusing an Affymetrix fluidics station and scanned on an Affymetrix scanner 3000with workstation. Images were processed and raw data were extracted withGeneChip Operating Software (GCOS).

1. Jiang X, Wang M, Graham DY, Estes MK: Expression, self-assembly, and antigenicity of the norwalk virus capsid protein. Journal of Virology 1992;66:6527-6532.

2. Green KY, Kapikian AZ, Valdesuso J, Sosnovtsev S, Treanor JJ, Lew JF: Expression and self-assembly of recombinant capsid protein from the antigenically distinct hawaii human calicivirus. Journal of Clinical Microbiology 1997;35:1909-1914.

3. Baric RS, Yount B, Lindesmith L, Harrington PR, Greene SR, Tseng FC, Davis N, Johnston RE, Klapper DG, Moe CL: Expression and self-assembly of norwalk virus capsid protein from venezuelan equine encephalitis virus replicons. Journal of Virology 2002;76:3023-3030.

4. Harrington PR, Yount B, Johnston RE, Davis N, Moe C, Baric RS: Systemic, mucosal, and heterotypic immune induction in mice inoculated with venezuelan equine encephalitis replicons expressing norwalk virus-like particles. Journal of Virology 2002;76:730-742.

5. Xia M, Farkas T, Jiang X: Norovirus capsid protein expressed in yeast forms virus-like particles and stimulates systemic and mucosal immunity in mice following an oral administration of raw yeast extracts. Journal of Medical Virology 2007;79:74-83.

6. Tacket CO, Sztein MB, Losonsky GA, Wasserman SS, Estes MK: Humoral, mucosal, and cellular immune responses to oral norwalk virus-like particles in volunteers. Clinical Immunology 2003;108:241-247.

7. LoBue AD, Lindesmith L, Yount B, Harrington PR, Thompson JM, Johnston RE, Moe CL, Baric RS: Multivalent norovirus vaccines induce strong mucosal and systemic blocking antibodies against multiple strains. Vaccine 2006;24:5220-5234.

8. Nicollier-Jamot B, Ogier A, Piroth L, Pothier P, Kohli E: Recombinant virus-like particles of a norovirus (genogroup ii strain) administered intranasally and orally with mucosal adjuvants lt and lt(r192g) in balb/c mice induce specific humoral and cellular th1/th2-like immune responses. Vaccine 2004;22:1079-1086.

Noroviruses (NoV), belong to the family of caliciviridae, are the leading cause of outbreaks of non-bacterial gastroenteritis both in the U.S. and worldwide. Currently, a vaccine is not available but certain high-risk groups, e.g. food handlers, health care workers, the military and probably the old people, are in a highly demand of norovirus vaccine. NoV genome is divided into three open reading frames (ORFs), ORF1, ORF2 and ORF3, in which ORF2 encodes norovirus capsid protein. Expression of ORF2 in baculovirus [1,2], Venezuelan equine encephalitis virus (VEE) [3,4] and Yeast [5], respectively, is capable to form virus-like particles (VLPs) that are antigenically and morphologically similar to native whole norovirus but without viral RNA, and both monovalent and multivalent VLPs could elicit systemic and mucosal immune response in human volunteers [6] and in mice [7]. Furthermore, one study observed a Th1/Th2-like cellular response in mice induced by genogroup II norovirus VLPs [8]. In order to better understand the importance of ORF2 as norovirus vaccine candidate, we transfected Norwalk virus ORF2 plasmid DNA into a human mammalian intestinal cell line Caco-2. The whole genome expression profiles in ORF2 and vector alone-transfected cells were compared in triplicate experiments using an Affymetrix human U133_ 2.0 array.

Microarray analysisThe Affymetrix CEL files were imported into GeneSpring 7.3 and normalized by GC-Robust Multichip Average (GCRMA). Firstly we removed genes with absent signal, and filtered out genes with present or marginal signal and with at least two-fold increased or decreased mRNA expression between ORF2-transfected and vector only transfected cells. Genes with statistically different expression between ORF2-transfected and vector only transfected cells (p<0.05) were identified by one way ANOVA analysis. Average-linkage hierarchical clustering was carried out to cluster the distinct patterns in gene expression and the relationships between the samples. Genes identified by GeneSpring were uploaded into the Ingenuity Pathways Analysis tool to further analyze the gene ontology, including biological processes, cellular components, molecular functions and genetic networks.

Pengbo Liu, Ph.D.Rollins School of Public HealthEmory University1518 Clifton Road, L36Atlanta, GA 30322Phone: (404)727-9218Email: pliu5@sph.emory.edu

METHODS

INTRODUCTION

RESULTS

SUMMARY & CONCLUSIONS

ABSTRACT

REFERENCES

Fig. 3. Pathways that 2-fold changed geneswere involved in. The x-axis displays pathways that 2-fold altered genes involved in; Y-axis displays the ratio of genes in a given pathwaythat were 2-fold change divided by totalnumbers of genes that make up that pathway.

ORF2 Vector

• ORF2 plasmid DNA transfection into human mammalian Caco-2 cells resulted in a significant ORF2 mRNA expression from triplicate experiments.

• A total of 761 transcripts exhibited at least 2-fold differences and 54 transcripts were statistically significant different between ORF2-transfected and vector only-transfected cells.

• Hierarchical clustering analysis could clearly divide the expression profiles of ORF2 and vector alone transfected samples into two distinct groups, while could also distinguish the 134 statistically significant different genes into various clusters.

• ORF2 transfection resulted in numbers of 2-fold changed genes that are involved in several immunological pathways, e.g. antigen presentation pathway, interferon signaling, IGF-1(insulin-like growth factor 1) signaling, etc.

• FUT4 (fucosyltransferase 4), FUT1, TNFSF15 (tumor necrosis factor superfamily member 15), SLC4A4 (solute carrier family 4, member 4) are 2-fold changed genes that involved in the gastrointestinal diseases.

ACKNOWLEDGEMENT

This study was supported by Emory University Research Committee (URC). We are grateful to Dr. Kim Green at NIH for her kind gift of the Norwalk virus ORF2 clone.

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