1

Molecular Characterization of in vivo Adjuvant Activity in Influenza-1

Vaccinated Ferrets 2

3

Running title: Adjuvant activity in seasonal flu vaccination 4

5

Yuan Fang1,2,3

, Thomas Rowe1,2

, Alberto J Leon1, David Banner

2, Ali Danesh

2,3, Luoling Xu

2, 6

Longsi Ran2, Steven E. Bosinger

6, Yi Guan

5, Honglin Chen

5, Cheryl C. Cameron

2, Mark J. 7

Cameron2, David J. Kelvin

1,2,3 4 * 8

9 1 Division of Immunology, International Institute of Infection and Immunity, Shantou University 10

Medical College, 22 Xinling Road, Shantou, Guangdong, P.R. China, 515041. 11

12 2 Division of Experimental Therapeutics, Toronto General Research Institute, University Health 13

Network, 101 College Street, Toronto, Ontario, Canada, M5G 1L7. 14

15 3

Department of Immunology, University of Toronto, Toronto, Ontario, Canada. 16

17 4 University di Sassari, Dipartimento di Scienze Biomediche, Sassari,, Italy 18

19 5 Division of Virology, International Institute of Infection and Immunity, Shantou University 20

Medical College, 22 Xinling Road, Shantou, Guangdong, P.R. China, 515041. 21

22 6

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, 23

Pennsylvania, USA. 24

25

*Corresponding author Dr. David J. Kelvin, Division of Immunology, International Institute of 26

Infection and Immunity, Shantou University Medical College, 22 Xinling Road, Shantou, 27

Guangdong, P. R. China 515041. 28

Email: dkelvin@uhnresearch.ca; Tel and Fax: (+86)-754-88573991 29

30

Word count for the abstract: 181 31

Word count for the text (except reference, figure legends and table footnotes): 7,285 32

Abbreviation used: 33

ODN, oligodeoxynucleotide; PEG-IFN, pegylated interferon-alpha2b; CFA, Complete Freund’s 34

adjuvant; ISG: interferon stimulated gene; TLR, toll-like receptor; IRF, interferon regulatory 35

factor. 36

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.02305-09 JVI Accepts, published online ahead of print on 9 June 2010

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ABSTRACT 37

The 2009 H1N1 influenza pandemic has prompted a significant need for the development 38

of efficient, single-dose adjuvanted vaccines. Here we investigated the adjuvant potential of CpG 39

Oligodeoxynucleotide (ODN) when used with human seasonal flu vaccine in ferrets. We found 40

that CpG ODN-adjuvanted vaccine effectively increased antibody production and activated type 41

I interferon (IFN) responses, compared to vaccine alone. Based on these findings, pegylated IFN-42

α2b (PEG-IFN) was also evaluated as an adjuvant in comparison to CpG ODN and Complete 43

Freund’s Adjuvant (CFA). Our results showed that all three adjuvant-added vaccines prevented 44

seasonal human H1N1 Brisbane59 virus replication more effectively than vaccine alone. Gene 45

expression profiles indicated that, as well as up-regulating IFN-stimulated genes (ISGs), CpG 46

ODN enhanced B cell activation and increased TLR4 and IRF4 expression, whereas PEG-IFN 47

augmented adaptive immunity by inducing MHC transcription and Ras signaling. In contrast, 48

using CFA as an adjuvant induced limited ISG expression but increased the transcription of 49

MHC, cell adhesion molecules, and B cell activation markers. Taken together, our results better 50

characterize the specific molecular pathways leading to adjuvant activity in different adjuvants-51

mediated flu vaccination. 52

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INTRODUCTION 53

Influenza infection is a prominent threat to human health around the world and can cause 54

severe morbidity and mortality in susceptible individuals due to acute respiratory disease. 55

Among the approaches to limit severe illness caused by influenza, vaccination is a critical 56

component in the prevention of the spread of infection. The human seasonal influenza vaccine 57

usually includes antigens from different influenza subtypes, H1N1, H3N2 and influenza type B, 58

which are predicted to circulate in the following flu season. However, this vaccine provides 59

protection to only 75% of the vaccinated population (15), and the protection efficacy in 60

immunized elderly individuals is lower than 50% (28). 61

For over 70 years, adjuvants have been used to enhance antigen-specific immune 62

responses. CpG ODN and type I IFN have been evaluated for their efficacy in commercial 63

influenza vaccines (4,53) with the general conclusion that adjuvant-mediated vaccines induce 64

stronger antibody responses and elevated cytotoxic T lymphocyte (CTL) activity. CpG ODN is a 65

ligand of toll-like receptor (TLR) 9, which is mainly expressed by plasmacytoid dentritic cells 66

(pDC), lymphocytes B and monocyte/macrophages. TLR9 stimulation by CpG can effectively 67

induce type I IFN responses and augment humoral responses (34,57). Type I IFN signaling is 68

thought to be critical to the initiation of innate immune responses to viral infections, and the IFN 69

stimulated genes (ISG), which include a variety of transcription factors, cytokines and 70

chemokines, appear to be involved in stimulating adaptive immunity and eliminating the virus 71

from the host (65). As an adjuvant, type I IFN has been shown to induce higher CTL 72

proliferation and antibody secretion than alum, and was equal to Complete Freund’s Adjuvant 73

(CFA), considered to be the gold standard adjuvant for use animal models (53). However, the 74

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connection between gene regulated immune protection and adjuvant-mediated vaccination are 75

still unknown. 76

In this study, the domestic ferret (Mustela putorius furo), a well-established model to 77

study the pathogenicity of influenza virus (42), was used to better characterize adjuvant activity 78

following influenza vaccination. CpG-adjuvanted influenza vaccination resulted in an increased 79

antibody response compared to vaccine alone in ferrets. In addition, elevated ISG mRNA levels 80

were observed at an early stage post immunization, presumably in part due to IFN-α signaling 81

activation. Based on these results and the known activities of type I IFN (38,65), it was expected 82

that type I IFN could also be an effective adjuvant in influenza vaccination. Pegylated IFN-α2b 83

(PEG-IFN), which has been approved for use in humans since 2001, was therefore tested for its 84

adjuvant potential in flu vaccination in ferrets along with CFA, which activates innate immune 85

responses partly through NOD-like receptor family member NOD2 (20,27), to compare its 86

effects on immune outcome and gene regulation to those of CpG and PEG-IFN. 87

MATERIALS AND METHODS 88

Animals 89

Male ferrets 4-6 months old were purchased from Marshall Bioresources (New York 90

city, NY, USA) and maintained at the Animal Resources Centre (University Health Network, 91

Toronto, Canada). Ferrets were routinely screened for influenza infection. 92

Immunization 93

The 2007-2008 seasonal human flu vaccine, Fluviral (ID Biomedical Corporation of 94

Quebec), which contains 15 µg HA of each inactivated influenza virus strain, A/Solomon 95

Islands/3/2006 (H1N1), A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004 in 0.5ml, was 96

used. Fluviral vaccine (100 µl) was mixed with 100 µl of total 500 µg class B CpG ODN (10104, 97

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Coleypharma, USA) four hours before injection. Each group of ferrets was injected with either 98

200 µl PBS, 100 µl PBS plus 100 µl Fluviral or 200 µl CpG added Vaccine. At Day 35 following 99

primary immunization, all ferret groups were boosted with Fluviral vaccine alone. All the 100

animals were vaccinated through intramuscular (i.m.) injection. Fluviral vaccine was abbreviated 101

as V2007

and the Fluviral vaccine administered with PBS and CpG was abbreviated as V2007

-102

Alone and V2007

+ CpG in the text. 103

The 2008-2009 seasonal human flu vaccine Vaxigrip (Sanofi Pasteur Limited), which 104

contains A/Brisbane/59/2007 (H1N1), A/Brisbane/10/2007 (H3N2) and B/Florida/4/2006, was 105

mixed with 100 µl PBS containing 1 µg PEG-IFN-α2b and 100 µl CFA before the injection. 106

Ferrets were also vaccinated intramuscularly with CpG added vaccine, vaccine alone, and PBS. 107

The abbreviations of the vaccinations were recorded as V2008

-Alone, V2008

+ CpG, V2008

+ IFN 108

and V2008

+ CFA. The amount of CpG and CFA were chosen based on the previous studies 109

which showed optimal adjuvanticity with various antigens (50,68). The dose of PEG-IFN 110

(1µµµµg/kg) was chosen according to the manufacture’s recommendations for use of PEG-IFN 111

in humans (UNITRON PEG, Schering-Plough). 112

ELISA for Anti-Influenza antibodies 113

Serum from PBS, V2007

-Alone and V2007

+ CpG injected ferrets was collected at Days 0, 114

14, 21, 28, and 35 after primary immunization and at Day 7 post second vaccine injection. 115

Antibody responses were assessed by ELISA assay. Briefly, ELISA plates were directly coated 116

with 2007-2008 FLUVIRAL vaccine at 5 µg/ml overnight at room temperature. Plates were 117

washed with PBS containing 0.05% Tween 20 (T-PBS) and blocked with 1% bovine serum 118

albumin (BSA) for one hour at 37°C. Antigen-coated plates were washed with T-PBS and 119

incubated with 1:1,000 diluted serum samples overnight at 4°C. After washing with T-PBS, 120

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plates were incubated with goat anti-ferret immunoglobulin (IgM and IgG) HRP conjugates 121

(Rockland Immunochemicals, Gilbertsville, USA) in a 1:10,000 dilution for 2 hours at 37°C. The 122

reaction was developed by o-phenylenediamine for 30 minutes and the optical density was read 123

at 450nm. 124

Haemagglutination Inhibition (HI) Test 125

Ferret serum samples were treated with receptor destroying enzyme (RDE) at 37°C 126

overnight. Fresh turkey red blood cells (TRBC) were washed and diluted in PBS to a 127

concentration of 0.5% (v/v). The non-immunized and immunized ferret sera were serially diluted 128

in PBS in 96-well V bottom cell culture plates. The serial diluted sera from PBS, V2007 + PBS 129

and V2007 + CpG groups were incubated with 25 µl (8HA units/50 µl) of A/Solomon Islands/3, 130

A/Wisconsin/67/2005 and B/Malaysia/2507/2004 strains (CDC at Atlanta, Georgia, USA) 131

separately for 15 minutes. Then, 50 µl of 0.5% TRBC was added and the plates were incubated 132

at room temperature for 30 minutes. The HI titer was the reciprocal of the highest serum dilution 133

to completely prevent agglutination. The same assay was applied on the 2008-2009 vaccine 134

immunized ferret serum by using 8 HA units/50 µl of A/Brisbane/59/2007 (H1N1) and 135

A/Brisbane/10/2007 viruses. 136

Microneutralization Assay 137

The serum neutralizing antibodies of the Fluviral and Vaxigrip immunized ferrets were 138

determined using the viruses A/Solomon Islands/3/2006 (H1N1) and A/Brisbane/59/2007 139

(H1N1) respectively for analysis by microneutralization (MN) assay described in Rowe et al., 140

1999 (58). Briefly, the 50% tissue culture infectious dose (TCID50) of each virus was determined 141

by titration in MDCK cells under biosafety level 2 conditions. The serially 2-fold diluted RDE 142

treated serum at a starting dilution of 1:10 was tested for neutralizing the 100 TCID50/50 µl of 143

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each virus in MDCK cell monolayer. The cytopathic effect was read after incubation for 20 144

hours. 145

Infection and Monitoring of Ferrets 146

All the immunized ferrets and PBS control group animals were moved at least 4 days 147

prior to infection to the BSL-2 animal holding area, where they were housed in cages contained 148

in bioclean portable laminar flow clean room enclosures (Lab Products, Seaford, Del.). Prior to 149

infection, baseline temperatures were measured twice daily for at least 3 days. Ferrets were 150

anesthetized with ketamine (25mg/kg), xylazine (2mg/kg), and atropine (0.05mg/kg) by the 151

intramuscular route and infected intranasally (i.n.) with a total of 1ml of 106 EID50 of virus/ml in 152

phosphate buffered saline (PBS) delivered to the nostrils. Temperatures were measured every 153

day by using a subcutaneous implantable temperature transponder (BioMedic Data Systems, Inc., 154

Seaford, Del.). Pre-infection values were averaged to obtain a baseline temperature for each 155

ferret. The change in temperature (°C) was calculated at each time point for each animal. Clinical 156

signs of sneezing (before anesthesia), inappetence, dyspnea, and level of activity were assessed 157

once daily. A scoring system based on Reuman et al.’s work (56) was used to assess the activity 158

level as follows: 0, alert and playful; 1, alert but playful only when stimulated; 2, alert but not 159

playful when stimulated; and 3, neither alert nor playful when stimulated. A relative inactivity 160

index was calculated as follows: Σ(day 1 to day 5) [score + 1]n/Σ(day 1 to day 5) n, where n equals the total 161

number of observations. A value of 1 was added to each base score so that a score of 0 could be 162

divided by a denominator, resulting in an index value of 1.0. 163

All the infected animals were euthanatized by intracardiac injection of Euthanasia V 164

solution (1ml/kg body weight) at Day 5 p.i. Tissues from nasal turbinates and major organs 165

including lung and spleen were collected either in Trizol or in formalin for later analyses. 166

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Collection of Nasal Wash and Virus Titration 167

Nasal washes were collected on Day 1, 2, 3 and 5 post infection (p.i.). Ferrets were 168

anesthetized as described above, and 0.5 ml of sterile PBS containing 1% bovine serum albumin 169

and penicillin (100U/ml), streptomycin (100µg/ml), and gentamicin (50µg/ml) was injected into 170

each nostril and collected in a petri dish when expelled by the ferret. The volume was brought up 171

to 1 ml with cold sterile PBS plus antibiotics. Sedated ferrets were weighed on Day 0 and Days 172

1, 2, 3, 4, 5 p.i. To determine the viral load in the nasal washes, 20 µl supernatant of nasal wash 173

was added to the 180 µl vDMEM (DMEM, 1% BSA, 50µg/ml gentamicin, 100 U/ml penicillin, 174

100 µg/ml streptomycin, 2.5µg/ml amphotericin B) cultured MDCK cells (2×105/ml) and 175

followed by a 10-fold dilution on a 96-well cell culture plate. After incubation for 2 hours, the 176

media was replaced with the 200 µl/well fresh vDMEM and the cells were cultured for 6 days. 177

On Day Six, 50 µl of the cultured media from each well was transferred to the V bottom 96-well 178

plate and 50 µl 0.5% TRBC was added to run a HI test. The virus titers expressed as TCID50/ml 179

from each nasal wash sample was calculated by Reed-Muench method. 180

Primer Design and Synthesis 181

Each gene primer was designed based on the conserved gene sequences obtained through 182

the alignments of the coding sequences from the various species of dog (Canis lupus), domestic 183

cat (Felis catus), human (Homo sapiens), cattle (Bos taurus) and/or pig (Sus scrofa). Primers 184

were designed specifically for detecting the ferret target genes including the paralog genes 185

of each family to produce a PCR product in the range of 60~250 base pairs (bp) using Primer3 186

software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The primers’ sequences 187

(Table 1) have GC content between 40-60% and penalties were set against self and pair 188

complements to deter primer-dimer formation. The primers were synthesized desalted under 189

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HPLC purity by Operon (Huntsville, AL) and Invitrogen (Shanghai, China). The ferret specific 190

gene primers available at ATCC BEI Resources (http://www.beiresources.org) are listed in Table 191

1. 192

Cloning and Sequencing of Partial Coding Sequences of Ferret Immune Genes 193

Polymerase chain reaction (PCR)-amplified products of target genes derived from 194

mitogen-activated ferret (Mustela putorius furo) PBMCs, splenocytes and lung tissue. Amplified 195

PCR products were cloned using the TOPO-TA cloning kit (Invitrogen, Burlington, ON) per the 196

manufacturer’s instructions. Sequencing was done by the laboratory at International Institute of 197

Infection and Immunity (Shantou, China). Sequences were analyzed using the BLASTN program 198

of the National Center for Biotechnology Information portal and deposited to the public database 199

GenBank (NCBI accession numbers are listed in Table 1). 200

Whole Blood Collection, RNA Extraction and cDNA Synthesis 201

Whole blood (1.5 ml) from each ferret was collected into a Paxgene tube (Qiagen, 202

Mississauga) at Day 1, 3 and 5 after first vaccination and Day 7 post second immunization. RNA 203

was extracted and purified by Qiagen Paxgene blood RNA kit according to the manufacturer’s 204

instructions. RNA quality and concentration were determined by a spectrophotometer 205

(Eppendorf, Mississauga, Canada). 500 ng of total RNA was reverse transcribed using 206

SuperScript II reverse transcriptase (Invitrogen, Burlington, ON) in a 20 µl reaction under the 207

following conditions: 6.25 µM random hexamers primer (Applied Biosystems), 50 mM Tris-HCl 208

pH 8.3, 3 mM MgCl2, 75 mM KCl, 0.5 mM of dATP, dGTP, dTTP, and dCTP, 10 mM DTT, 40 209

U RNase Inhibitor (Applied Biosystems) and 200 U SuperScript II RNAse H- reverse 210

transcriptase at 42ºC for 1 hr. 211

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Real-time PCR 212

Quantitative real time PCR (QRT-PCR) was performed using an ABI-PRISM 7900HT 213

Sequence Detection System and SYBR green PCR Master Mix (Applied Biosystems, Foster 214

City, California). Each primer pair was tested with serially diluted concentrations of a control 215

cDNA to generate a standard curve. Samples and standards were analyzed in triplicate. Each 216

QRT-PCR reaction was performed a 10 µl reaction volume with 0.25 µl of cDNA, 1 µl primers 217

(500 nM each primer) and 5 µl of SYBR green PCR MasterMix in ABI-PRISM optical 384-well 218

plates. β-actin was used as the house-keeping gene for sample normalization. 219

Microarray analysis 220

Peripheral blood RNA isolated from each animal at day 1 post vaccination was analyzed 221

by microarrays in the 2008-2009 vaccine study (n=3/group). Briefly, cRNA was prepared from 222

500 ng total whole blood RNA by two-cycle cRNA synthesis according to the manufacturer’s 223

protocol (Affymetrix, Santa Clara, CA). cRNA samples (20 µg) were labeled and hybridized to 224

Affymetrix Canine 2.0 oligonucleotide arrays to monitor gene expression of over 18,000 Canis 225

familiaris mRNA/EST-based transcripts and over 20,000 non-redundant predicted genes. To 226

minimize batch effects, RNA extraction and cRNA preparation of all samples were 227

performed at the same time. Also, cRNA samples were hybridized on the same batch of 228

microarray chips. As we have established in previous studies (2,59), canine arrays were used 229

due to the high levels of homology between canine and ferret nucleotide sequences (see the 230

homology analysis for the current study in supplemental Table S1). 231

The arrays were scanned during the same session using an Affymetrix GCS3000 7G 232

system according to standard Affymetrix protocols. Probe-level analysis was performed using 233

Probe Logarithmic Error Intensity Estimate (PLIER). The raw intensity values for each 234

individual target on the Affymetrix chips were pre-processed with variance stabilization, log2 -235

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transformation and were normalized against the PBS control group datasets with ArrayAssist V 236

5.5.1 (Stratagene, USA). T-test was performed with Benjamini-Hochberg false discovery rate 237

(FDR) correction. Genes with a significant difference (p ≤ 0.05; fold change ≥ 1.5 or ≤ -1.5) 238

were selected for agglomerative hierarchical clustering with Pearson distance metrics and 239

average linkage distance measurements between clusters using MultiExperimental Viewer 4.1 240

(60). 241

Ingenuity Pathway Analysis (IPA) 5.0 software (Ingenuity Systems Inc., Redwood City, 242

CA) was used to annotate and organize the gene expression data into networks and pathways. 243

Statistics 244

One-way ANOVA was used for statistical analysis of the results represented in Fig. 4A 245

and Fig. 6. Other analyses used the Student’s t-test for comparing two independent populations. 246

Quantitative PCR results from 50 ferret immune-related genes were represented as heat map 247

charts generated by the software MultiExperimental Viewer 4.1. Bar graphs were generated by 248

SigmaPlot 8.0. 249

RESULTS 250

Humoral responses of CpG ODN-mediated immunization 251

We first investigated the effects of CpG ODN as an adjuvant for the 2007-2008 seasonal 252

human flu vaccine, Fluviral (V2007

) in ferrets. Fluviral contains 15µg of HA protein from the 253

following influenza virus strains: A/Solomon Islands/3/2006 (H1N1), A/Wisconsin/67/2005 254

(H3N2) and B/Malaysia/2506/2004. Ferret serum samples were collected at time points 255

following the primary immunization and vaccine alone boost. Immune activation was determined 256

by anti-vaccine IgM and IgG antibody serum levels based on ELISA assays. Serum samples 257

from V2007

+ CpG treated animals had significantly higher IgM levels at Day 14 after primary 258

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immunization (Fig. 1A, left panel), when compared to the IgM level of V2007

-Alone group. 259

Interestingly, there were also statistically higher levels of antigen specific IgG in V2007

+ CpG 260

sera than V2007

-Alone treated animals at all the time points after primary vaccination and also at 261

Day 7 post boost (Fig. 1A, right panel). These results suggest that CpG ODN mediated influenza 262

vaccination increases humoral immunity by inducing antibody production and promoting faster 263

antibody class switching. 264

To confirm the results of ELISA assays, the antibody titers were also assessed by 265

haemagglutination inhibition (HI) and microneutralization (MN) assays. HI assays indicated that 266

the sera from V2007

+ CpG treated animals contained significantly higher levels of antibody titers 267

than the V2007

-Alone treated animals (Fig. 1B) after primary vaccination to Day 7 post boost. The 268

MN assay also showed that the serum antibody from V2007

+ CpG treated animals had 269

significantly higher titers against the A/Solomon Islands/3/2006 (H1N1) virus than the animals 270

treated with vaccine alone at all the time points (Fig. 1C). These data further indicate that using 271

CpG as adjuvant is able to augment the humoral immune response to influenza vaccinations. 272

Regulation of ISGs by CpG adjuvanted vaccination 273

Having shown that the addition of CpG ODN to V2007

upregulated humoral responses 274

after vaccination, we assessed gene expression activity associated with increased 275

immunogenicity at mRNA level. Quantitative PCR (Q-PCR) was used to generate a 50-gene 276

expression profile from the whole blood RNA samples collected at Days 1, 3, and 5 after primary 277

immunization and Day 7 post boost in the vaccination groups V2007

+ CpG, V2007

-Alone and PBS 278

V2007

Boost. The PBS treated group was used as control for normalization at Days 1, 3 and 279

5 after primary vaccination and at Day 7 post boost. The identical gene expression pattern 280

was observed in CpG adjuvanted and non-adjuvanted vaccination groups following 281

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normalization to PBS group at day 0 (data not shown). Since most of the immune related 282

genes were significantly regulated at Day 1 in the V2007

+ CpG group compared to the control 283

group, the Day 1 Q-PCR data of 50 genes was illustrated by heat map (Fig. 2A). The expression 284

levels of the 12 immunologically relevant genes, from Day 1 after primary vaccination to Day 7 285

post boost, were statistically analyzed and represented by bar graphs (Fig. 2B, 3). 286

ISG molecules, such as the anti-viral genes OAS1, RIG-I, Mx-1, ISG15, ISG20; 287

transcription factors IRF1, IRF7, STAT1, STAT2; and the Th1 cell chemokine CXCL10 (54), 288

which are induced by type I IFN during the virus infection, were significantly elevated at Day 1 289

after primary vaccination (Fig. 2A). However, at later time points the expression level of most 290

ISGs decreased to basal level or became downregulated, such as OAS1, IRF7 and RIG-I. Only 291

CXCL10 and STAT1 showed sustained increased expression levels at Day 7 after boost (Fig. 292

2B). Although the ISGs were modulated similarly in the group without adjuvant, at Day 3 and 293

Day 5 post primary vaccination the induced level of genes such as OAS1, RIG-I, ISG15 and 294

STAT2 were markedly lower than with CpG (Fig. 2B). This observation suggested that CpG 295

stimulated a faster and stronger type I IFN response than that in the animals treated with vaccine 296

alone. 297

Given that CpG mediated vaccination strongly induced ISGs, we then determined 298

whether IFN regulated ISG expression in vaccinated animals. We examined the mRNA level of 299

IFN-α and IFN-γ by Q-PCR. IFN-α was elevated in the V2007

+ CpG group at Day 1 after 300

primary vaccination and Day 7 post boost, whereas, IFN-γ levels were not highly increased by 301

vaccination plus CpG (Fig. 3A). Even though V2007

-Alone immunization increased the IFN-α 302

level at Day 3 and Day 5, it was not statically significant when compared to the V2007

+ CpG 303

group (Fig. 3A). Several studies have reported that IFN-stimulated response repressors, such as 304

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IRF-2, ICSBP and IRF4/PIP, likely terminate ISG transcription following IFN induction 305

(18,48,69). In this study, IRF4 was highly upregulated in the V2007

+ CpG group at Day 5 after 306

primary vaccination and Day 7 post boost (Fig. 3B). Since ISG expression was low during these 307

time points, these results implied a negative role for IRF4 in the regulation of IFN-stimulated 308

genes during adjuvant-mediated vaccination. Since IRF4 is also involved in the B cell class 309

switch recombination and plasma cell differentiation (32,40,41), the elevated IRF4 expression in 310

V2007

+ CpG immunized animals may indicate a role in humoral immunogenicity regulation. 311

IFN-α and CFA-mediated vaccination 312

We next evaluated IFN-α as an adjuvant to human influenza vaccination to determine if 313

the CpG-induced IFN response was responsible for the induced humoral response. Pegylated 314

IFN-α (PEG-IFN) was tested as an adjuvant for the 2008-2009 human flu vaccine (V2008

), 315

Vaxigrip, in ferrets. The activity of pegylated IFN-α was evaluated by antibody titer 316

quantification, and compared to the response of CpG ODN (V2008

+ CpG), CFA (V2008

+ CFA), 317

vaccine alone (V2008

-Alone) and PBS control groups. To determine antibody titers, HI and 318

microneutralization assays were run using serum samples collected on Days 14, 21, 28 and 35 319

post V2008

immunizations. Quantification of HI titers for the live influenza A viruses 320

Brisbane/59/2007 H1N1 and Brisbane/10/2007 H3N2 determined that CpG, PEG-IFN and CFA 321

mediated vaccinations induced significantly higher titers than vaccine alone (Fig. 4A). 322

Furthermore, the microneutralization assay showed that V2008

+ CpG, V2008

+ IFN and V2008

+ 323

CFA immunized ferrets had higher titers of Brisbane/59/2007 H1N1 neutralizing antibody than 324

V2008

-Alone at Day 14 after vaccination (Fig. 4B). Later time points showed only CpG and CFA 325

mediated vaccinations stimulated statistically stronger antibody responses. In a further 326

comparison of antibody induction among the adjuvanted immunizations, both CpG and CFA 327

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adjuvanted vaccines stimulated significantly higher antibody titers than the PEG-IFN adjuvanted 328

vaccine. However, significant differences were not observed between the CpG and CFA groups. 329

These results indicate that adjuvant addition increases humoral responses and that V2008

+ CFA 330

produces the strongest responses of the three adjuvant-mediated vaccinations. 331

Virus infectivity in adjuvant-treated animals 332

We next determined whether an increase in humoral immunity led to increased protection 333

from viral infection. Ferrets were immunized with the following combinations: PBS, V2008

-334

Alone, V2008

+ CpG, V2008

+ IFN and V2008

+ CFA. At day 42 following immunization, the 335

ferrets were infected with 106 EID50 Brisbane/59/2007 H1N1 virus to evaluate the protection 336

efficacy of adjuvant-mediated vaccinations. Brisbane/59/2007 H1N1 virus has been found to 337

replicate only in the nasal cavity of ferrets (47,59). From Day 2 p.i., 50-75% of the animals in 338

each group exhibited clinical signs of respiratory disease, including nasal discharge and 339

sneezing. The symptoms lasted for 2-3 days and decreased at Day 5 p.i. All animals exhibited 340

increased temperature at Day 2 p.i., which diminished by Day 3, except the groups of V2008

-341

Alone, V2008

+ IFN and PBS, which showed a second increase in temperature on Day 4 p.i.. In 342

addition, the animals displayed a decrease in activity by Day 3 p.i., except the animals 343

immunized with V2008

+ CFA. The relative inactivity index (Table 2) showed that V2008

+ CFA 344

was 1.1, V2008

+ CpG and V2008

+ IFN were both 1.3, and V2008

-Alone and PBS control groups 345

were 2.0 and 1.6, respectively. These results suggest that adjuvant-mediated vaccination in 346

ferrets dramatically reduces influenza severity compared to receiving the vaccine alone. 347

To evaluate infectivity, nasal washes were collected from animals on each day post 348

infection and viral loads (TCID50/ml) were calculated. Although viral loads in each group were 349

elevated at Day 1 and Day 2 p.i., the virus replication in the nasal turbinates of the V2008

+ CFA 350

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group was significantly lower than in the PBS control group. Furthermore, the Day 3 p.i. virus 351

titer measured in nasal washes of adjuvant groups were markedly diminished compared to those 352

of the PBS control (Fig. 5), whereas no significant decrease was observed in the ferrets that 353

received vaccine alone. By Day 5 p.i., the virus titer was not detectable in the majority of 354

animals. Interestingly, the assessment of antibody titer in serum at Day 5 p.i. showed that 355

adjuvant-immunized animals had significantly higher antibody levels than animals treated with 356

vaccine alone, as determined by HI and neutralization assays (Fig. 6A, B). Additionally, in nasal 357

washes collected at day 5 p.i., the anti-Brisbane/59 IgG levels in the CpG and CFA adjuvanted 358

vaccination groups were significantly higher than the vaccine alone (data not shown). Given the 359

clinical results and the decreased viral loads and increased antibody production observed in 360

animals vaccinated with adjuvant compared to animals receiving the vaccine alone, we suggest 361

that adjuvant induces a faster and stronger memory antibody response upon infection. 362

Microarray analysis of CpG adjuvant immunization 363

Above we showed that the expression of our selected immune related genes changed 364

most significantly at Day 1 after vaccination with CpG compared to control (Fig. 2A). We next 365

investigated the large scale gene expression profile of Day 1 adjuvant vaccinations by microarray 366

analysis. The heat map (Fig. 7) represents the expression of immune-related genes significantly 367

altered at Day1 post vaccination from adjuvant-mediated V2008

vaccinations (one ferret per 368

column). A similar expression pattern was observed by Q-PCR for three genes selected from the 369

antigen presentation, adaptive immunity and complement clusters shown in Fig. 7 (see 370

supplemental Fig. S1). Fold gene expression changes for each adjuvant-treated group compared 371

to PBS controls are listed in Table 3. Also, we used vaccine alone as a comparator and 372

observed a similar expression pattern to that of the PBS group (data not shown). 373

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Consistent with our Q-PCR results of V2007

+ CpG vaccination, which showed 374

upregulation of 10 ISGs, the microarray data determined that 15 ISG members were increased by 375

at least 1.5 fold in V2008

+ CpG immunization (Fig. 8A, Table 3). The Ingenuity Pathway 376

Analysis (IPA), combining the Q-PCR and microarray data, suggests that CpG-adjuvanted 377

vaccination stimulates type I IFN signaling and activates ISG members through STAT1 and 378

STAT2 (Fig. 8B upper panel). Also, toll-like receptor 4 (TLR4), which specifically recognizes 379

bacterial lipopolysaccharides (LPS) and mediates innate immunity (31), was induced following 380

CpG-adjuvanted vaccination (Table 3). Transcription analyses showed that co-stimulatory 381

molecules and MHC genes were not elevated by vaccination with CpG, except for the 382

upregulation of cathepsin family antigen processing genes, CTSB and CTSS. Similarly, neither 383

microarray nor Q-PCR showed increased IFN-γ levels after CpG-mediated vaccination when 384

compared to the vaccine alone. Interestingly, the immune regulator SOCS1 (Table 3), which 385

negatively regulates IFN-γ signaling (10), was found to be upregulated in V2008

+ CpG 386

immunized animals by microarray analysis. 387

Microarray analysis of IFN-α adjuvant immunization 388

Given that CpG-mediated vaccination significantly increased ISGs, we tested IFN-α for 389

its direct adjuvant potential during immunization against influenza. We have found that 390

treatment with PEG-IFN alone resulted in significantly increased mRNA levels of ISGs in ferrets 391

at early time points (unpublished data). Here, microarray analysis revealed that 345 genes were 392

highly upregulated following V2008

+ IFN vaccination. Similar to the gene profile of CpG 393

mediated immunization, V2008

+ IFN stimulated the expression of ISGs, including STAT1, IRF2, 394

OAS1/2, ISG15, USP18, IFIT2, RSAD2, IFI44 and IFI44L (Fig. 8A), although to a lesser extent 395

than the V2008

+ CpG group, shown by the IPA analysis (Fig. 8B lower panel). 396

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V2008

+ IFN immunization also upregulated antigen processing and presentation genes, 397

such as CTSD and MHC class Ib, as well as the T cell adhesion molecule, CD84 (66) (Table 3). 398

We also found the MHC enhanceosome member RFX5, which regulates MHC during antigen 399

presentation (55), was significantly increased following PEG-IFN mediated vaccination. 400

Interestingly, PEG-IFN mediated vaccination also induced the gene expression of intracellular 401

signaling molecules Ras and the MEK member MAP2K2 (Fig. 8C), which are known mediators 402

of B cell proliferation and B cell memory after CD40 stimulation (7,8). JUNB, which is 403

downstream of Ras following BCR stimulation (70), was also up-regulated in V2008

+ IFN 404

vaccination. Additionally, the expression of the guanine nucleotide activating protein (GAP), 405

RGS1, involved in B cell activation (26), which was elevated in the V2008

+ CpG group, was also 406

increased in V2008

+ IFN immunized ferrets (Table 3). 407

Microarray analysis of CFA adjuvant immunization 408

The precise molecular mechanism of CFA-mediated influenza immunization is not 409

completely understood. We therefore investigated the gene profile of CFA-adjuvanted influenza 410

vaccination in ferrets. Microarray analysis revealed that a total of 1,255 genes were regulated at 411

Day 1 post V2008

+ CFA immunization. Since CFA is thought to contain the NOD2 agonist 412

MDP, which induces NF-κB and MAPKs to initiate proinflammatory cytokine expression in 413

innate immune cells (36), we examined the expression of NOD2 signaling genes. The expression 414

of the MAP Kinase family member ERK was induced at Day 1 after CFA mediated vaccination 415

(Table 3), but proinflammatory cytokines were not induced. 416

CFA may also contain a TLR ligand, which could synergistically activate DCs with MDP 417

(19,27). Similar to CpG- and IFN-mediated vaccination, the expression of antigen processing 418

genes, namely CTSD, MHC class I DLA-64, MHC class II HLA-DMA/CLIP (CD74) and MHC 419

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transcription enhancesome RFX5 (Fig. 7, Table 3) were significantly increased in CFA-mediated 420

immunization. However, the MHC class II transactivator (CIITA), which regulates MHC class II 421

gene transcription, was significantly down-regulated (Table 3). We also found that CFA 422

activated the expression of several adhesion molecules, including Integrin-β, VCAM1, BCAM, 423

CD36, CD84 and CD44 (Fig. 9A), and genes such as Zyxin, Talin 1, CRKL, RAP2A and Rho 424

family members involved in integrin signaling and mediating cell adhesion and motility (Fig. 425

9B). Moreover, expression of CXCL14, a chemokine involved in B cell migration and activation 426

(63), was also found elevated (Table 3). 427

B cell intracellular signaling molecules, such as Lyn, Syk, and PI3K, were significantly 428

up-regulated by CFA at Day 1 (Fig. 9A, C). In addition, increased mRNA level of B cell 429

proliferation regulator Bam32 (22,43) was observed in CFA-mediated vacination. In contrast to 430

the PEG-IFN-induced transcription of MEK member MAP2K2, involved in Ras mediated BCR 431

signaling, CFA activated the expression of Ras downstream effectors, c-Raf and MAP Kinase 432

ERK (Fig. 9C). Following BCR engagement, Ras activation is one result of Ca2+

signaling (9) 433

which leads to gene induction through the calcineurin-NFAT pathway to stimulate B cell 434

proliferation (23). Indeed, the calcineurin family members PPP3R1, NFAT molecule NFATC3, 435

and the calcineurin-NFAT activation regulators G protein complex (GNA15, GNB3, GNG2 and 436

GNG11) and CK1 (CSNK1D and CSNK1G2), were all highly induced following CFA-437

adjuvanted vaccination (Fig. 9D). Moreover, we found that the small G protein Rac, another 438

factor which activates NF-κB and JNK signaling (6,25,45), was induced after vaccination with 439

CFA (Fig. 9C) and AICDA (AID), essential for initiating the immunoglobulin gene 440

hypermutation (SHM) and class switch recombination (CSR) in B cells (3), was increased by 2.6 441

fold in CFA mediated vaccination (Table 3). Although AID was induced, only one of its 442

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activator molecules, Oct2 (51), was significantly increased whereas the other, HoxC4, was 443

downregulated (Fig. 9C). 444

Lastly, SOCS family member, SOCS3, was induced by V2008

+ CFA treatment (Table 3). 445

High SOCS3 expression may favour Th2 cell differentiation and inhibit IL12-mediated Th1 446

development (62), which is consistent with our finding that IL12A (Table 3) was significantly 447

down-regulated in CFA-adjuvanted vaccination. Also, more complement components, such as 448

C1QL2, C1R, C5AR1 and CFI (Fig. 7, Table 3), were activated in V2008

+ CFA immunization 449

compared with the other adjuvanted immunizations which upregulated only FCN1 and C3 450

precursor. However, V2008

+ CFA stimulated few ISGs, such as transcription regulator IRF1, 451

IRF2, and antiviral gene RSAD2 (Table 3), when compared to CpG and PEG-IFN adjuvanted 452

vaccinations. 453

DISCUSSION 454

Here we investigated the adjuvant potential of CpG ODN when added to the seasonal 455

human influenza vaccine, and subsequently characterized the molecular gene signatures of the 456

induced immune responses. Since CpG ODN initiated robust IFN responses following 457

vaccination, we evaluated type I IFN as an adjuvant by using PEG-IFN in combination with the 458

human influenza vaccine, Vaxigrip. We also compared the effect of PEG-IFN to the standard 459

adjuvant, CFA. To demonstrate the activity of each adjuvant during immunization, gene 460

expression profiling was performed by microarray. 461

Gene expression profile generated by Affymetrix canine arrays was used to expand 462

on the gene signatures identified with Q-PCR analysis (Fig. 2). At this time, it is not known 463

whether microvariation in the canine probes affects detection of ferret orthologs. Our 464

group has established the utility of canine platform to assess ferret gene expression using a 465

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homology analysis of the limited publicly available ferret and canine cDNA sequences and 466

a ferret-specific Q-PCR validation strategy (2,59). Cross-species microarray analyses are 467

supported by previous studies (11,30,49), however, a lack of publicly available canine whole 468

blood microarray datasets has not allowed us to make the same comparisons. Nonetheless, 469

high homology was identified between numerous ferret and canine genes derived from the 470

current study (Table S1) and extensive ferret-specific Q-PCR validation on surrogate genes 471

was performed. 472

The CpG experiment showed that our genes of interest changed most significantly at day 473

1 following vaccination (Fig. 2), therefore this time point was chosen for more extensive 474

microarray analyses. We demonstrated that although CpG ODN led to the activation of ISGs and 475

subsequent stimulation of humoral responses, when PEG-IFN was used it was unable to 476

reproduce the humoral activation to the same extent as that of CpG ODN. Furthermore, CpG 477

ODN as well as IFN and CFA adjuvant-mediated immunizations stimulated stronger antibody 478

responses than the vaccine alone and gave better protection for animals following seasonal H1N1 479

infection. The microarray analysis for each adjuvant showed differing molecular signatures 480

indicating that distinct molecular pathways were activated depending on the adjuvant used. 481

Based on microarray profiling and Q-PCR analysis, the CpG-adjuvanted immunization 482

activated canonical type I IFN signaling responses. The upregulation of ISGs included the virus 483

sensing RIG-I and PKR, the virus replication inhibitors OAS1, 2 and L, the transcription factors 484

STAT1, STAT2, the IFN-regulating molecules IRF1, IRF7, and the Th1 cell chemoattractant 485

CXCL10. In plasmacytoid DC (pDC), CpG binds to the intracellular receptor TLR9 to activate 486

the IFN-α expression, which initiates the ISG stimulation (21). In our study, the ISG induction 487

was transient after vaccination and the relatively increased IFN-α expression was only observed 488

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at 24 hours post vaccination. Typically, pDCs are thought to be the primary producers of type I 489

IFN and that production of IFN following CpG stimulation leads to maturation of the 490

conventional DCs (cDCs), which in turn augments the B cell response toward a Th1-like 491

phenotype by inducing IFN-γ and IL12 (33). Our gene profiling did not support this mechanism 492

for CpG-induced pDC and cDC-mediated enhancement of humoral response through Th1 493

regulation, since molecular signatures associated with DC maturation and/or Th1 signaling were 494

not observed. Moreover, we found that SOCS1, the suppressor of IFN-γ signaling (10), was 495

significantly up-regulated. Therefore, CpG may function as a non-Th1 biasing adjuvant during 496

immunization, a possibility we are studying further. 497

Several recent studies indicate that CpG can directly activate B cells through TLR9 and 498

MyD88 to promote class switching toward a Th1 phenotype (29,39). In addition, TLR9 signaling 499

has been implicated in plasma cell proliferation and differentiation after immunization with 500

antigen and non-soluble CpG, which leads to IL-6 and IgM secretion (14). Here we found that 501

IRF4, which is involved in antibody class-switching and plasma cell differentiation (17,32,41), 502

was upregulated in CpG-mediated vaccination. Since both ligation of TLR9 and intracellular 503

activation of IRF4 regulate antibody class switching, we postulated that the IRF4 could be 504

induced downstream of TLR9 engagement in B cells. In this scenario, it is possible that 505

downstream activation of IRF4 may occur via MyD88-mediated NF-κB and/or AP-1 activation, 506

which has been a proposed route for B cell activation, proliferation and Ig production after TLR9 507

anchoring (52). High levels of IRF4 occurring after the antigen boost also suggests that IRF4 508

could play a role in the memory plasma cell differentiation, which is consistent with the findings 509

in the study by Klein et al. (32). Taken together, our data suggests that CpG-adjuvanted 510

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vaccination activates B cells via TLR9-mediated expression of genes such as IRF4, involved in 511

plasma cell differentiation and antibody class switching. 512

Interestingly, the microarray profiling of CpG mediated vaccination indicated that TLR9 513

activation correlated with TLR4 upregulation. TLR4 cooperates in BCR signaling to enhance the 514

antibody response through LPS ligation (13,46). Although LPS is the classic antigen for TLR4, 515

an updated list asserts that other pathogens, such as viral protein and parasitic heat shock 516

proteins, also bind to this innate receptor (1). Thus, TLR4 may be also involved in immune 517

activation by recognizing the vaccine antigen in conjunction with TLR9-mediated vaccine 518

responses. 519

As discussed above, type I IFN induces adaptive immunity by stimulating DC cell 520

surface co-stimulatory molecules and MHC antigens which enable DCs to activate B cells (37). 521

Since type I IFN is an inducer of adaptive immunity, we used pegylated IFN-α2b as an adjuvant 522

to compare with CpG-adjuvanted immunization. PEG-IFN induced similar ISG expression to 523

CpG-adjuvanted vaccination as determined by microarray analysis (Fig. 8 b). The MHC class I 524

gene was also induced possibly as a result of increased levels of the MHC transcription 525

enhancesome RFX5. An increase of the MHC class I molecule on the surface of antigen 526

presenting cells (APCs) can then activate T cells to express the adhesion molecule CD84. Since 527

RFX5 was also up-regulated in CFA adjuvanted vaccination, it is possible that RFX5 was 528

activated by the same signaling pathway and may represent as a common mediator of adjuvant 529

activity in both PEG-IFN and CFA mediated vaccination. In the future, it will be important to 530

elucidate the mechanism of RFX5 induction since MHC upregulation in our adjuvant-mediated 531

studies are important components of adaptive immunity. Interestingly, PEG-IFN stimulated Ras 532

and MEK genes involved in Ras-MEK-ERK signaling. Ras-MEK-ERK signaling in B cell plays 533

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an important role in generating the high affinity antibody (61). It is therefore likely that type I 534

IFNs can directly activate B cells through type I IFN receptor to initiate the Ras-MEK-ERK 535

pathway, as type I IFNs can stimulate B cells directly to produce antibody and express IFIT2/3 536

early after influenza infection (5,61). 537

Our microarray data suggest that IFN-α2b activates MHC class I expression which may 538

play a role in activating CD8+ T cells post-vaccination. Furthermore, the data implies that IFN-α 539

activates antibody generation by B cells through Ras signaling. However, our HI and 540

microneutralization assays showed antibody levels induced by PEG-IFN were lower than those 541

stimulated by CpG (Fig. 2). This decreased antibody production may be dose-dependent since 542

previous reports have shown that the Ig subclass was highly increased at a high dose of IFN-α 543

adjuvant administration (67); or possibly the IFN-α2b subtype could not fully activate the DC or 544

B cell, since IFN-α1 is the main subtype produced by pDC after virus infection. 545

CFA has been used for decades in animal models to generate high levels of antibody 546

against antigens. It is known that MDP, the NOD2 ligand, is the minimal essential component in 547

CFA (16). Unlike previous findings (36), the results of our microarray profiling in ferrets showed 548

that CFA-mediated vaccination did not stimulate a proinflammatory milieu but instead only 549

showed the elevation of the MAPK pathway gene ERK. Weak proinflammatory cytokine 550

responses, e.g. absence of IFN-γ gene expression, may be the result of a low dose of CFA 551

treatment (100 µµµµl, one injection) given to the ferrets. Even though a robust inflammatory 552

response was not observed, the transcription enhancesome RFX5 was upregulated upon CFA-553

adjuvanted vaccination along with MHC gene induction, which is associated with DC maturation 554

(44). Previous reports have shown strong synergism between TLR ligands and MDP when 555

administered together (64). Therefore, it is possible that there may be another ligand in CFA that 556

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contributes to the adjuvant activity. In particular, the TLR4 ligand can increase the maturation 557

potential of MDP on human DC by inducing co-stimulatory molecules and the MHC class II 558

gene (19). Furthermore, NOD2/TLR mediated MHC upregulation may be regulated by the 559

induced SOCS3, which is involved in the negative regulation of STAT3 and associated with 560

Th2-type signaling in DC (35). This is supported by our finding that the Th1-directing 561

cytokine IL12A (Table 3) was significantly down-regulated in CFA-adjuvanted 562

vaccination. 563

Mature DCs efficiently present antigens on the cell surface and directly promote 564

expression of B cell high-affinity receptors. This results in strong BCR signaling and subsequent 565

interaction with T helper cells to initiate antibody secretion cells and memory B cells (12). In this 566

study, we found that several important genes involved in B cell activation were induced in CFA 567

mediated vaccination, in particular, the molecules involved in Lyn-Syk-PI3K signaling pathway, 568

calcineurin-NFAT pathway and Ras-MEK-ERK pathway. Furthermore, our pathway analysis 569

suggested that B cell activation may have induced Calcineurin-NFAT, Ras-MEK-ERK and NF-570

κB signaling (24). These pathways in turn initiate the transcription of genes involved in B cell 571

expansion, plasma cell differentiation and antibody production, such as Bam32 (22), and Oct2 572

(POU2F2), which binds to the AICDA promoter and activates AID transcription (51) for 573

determining the antibody SHM and CSR (3). In addition, the molecules engaged in cell adhesion 574

and motility, complement components, and chemoattractant were highly stimulated in CFA 575

adjuvanted vaccination. 576

In contrast to type I IFN activation by CpG ODN or PEG-IFN, CFA stimulated a 577

relatively low level of ISG expression, which is unlike previously published findings that robust 578

type I IFN activation is a hallmark of CFA activity in mouse models (37). Given that CFA was 579

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administered with vaccine intramuscularly, the slow release of this emulsion from the injection 580

site to the peripheral circulation, or the more localized activity of this adjuvant, may result in the 581

limited ISG stimulation in peripheral blood at 24 hours post vaccination. It will be important to 582

evaluate gene regulation in the peripheral blood at later time points following CFA adjuvanted 583

vaccination to demonstrate the role of IFN responses in the adjuvanticity of CFA. 584

Altogether, our data suggests that the low dose of CFA activates the expression of MHC 585

molecules which are associated with DC activation through NOD2 and/or TLR signaling rather 586

than type I IFN receptor ligation. We contend that the activated DC may strongly activate BCR 587

signaling to initiate the B cell proliferation and plasma cell differentiation through high-Ca2+

-588

induced NFAT, ERK and NF-κB-regulated transcription. Given the paucity of ferret-specific 589

reagents, we could not isolate DC in this study to investigate this potential mechanism further. 590

Also, we were limited to one time point of gene expression profiling and cannot rule out 591

that some of the expression differences may be affected by different gene expression 592

kinetics or different dose of adjuvants. 593

In conclusion, we have identified both common and disparate signaling pathways 594

activated downstream of in vivo adjuvant activity during vaccination. Of note, RFX5 was a 595

common transcript induced by both IFN-α and CFA-adjuvanted vaccinations, representing a 596

focal point of adjuvant activity. Additionally, the identified signature molecules in our study 597

could be specifically targeted in future vaccines, thereby facilitating the efficacy of vaccination 598

and the development of host immunogenicity. 599

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ACKNOWLEDGEMENTS 600

We thank Dr. Alexander Klimov (CDC at Atlanta, USA) for the kind gift of the 2007-601

2008 human seasonal influenza viruses, and Lixia Guo and Zujiang Li at Shantou University 602

Medical College (Shantou, China) for assisting with ferret gene cloning. We would also like to 603

thank Jean Flanagan and Roman Skybin, from Animal Resources Centre at Toronto General 604

Hospital (Toronto, Canada), for the collection of ferret samples. We would like to give special 605

thanks to Dr. Alyson Kelvin for reviewing the manuscript. 606

This work is supported by grants from the Li Ka Shing Foundation, the Canadian 607

Institute of Health Research, Sardegna Ricerche and the National Institutes of Health. 608

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814

815

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Figure 1. CpG ODN-assisted vaccination increased influenza virus-specific antibody levels 816

in serum from immunized ferrets. 817

Influenza virus-specific antibody levels in serum from immunized ferrets were assessed by (A) 818

ELISA, (B) HA inhibition and (C) microneutralization assays. (A) Serum IgM (left panel) and 819

IgG (right panel) antibody levels against the commercial vaccine FLUVIRAL were measured at 820

Days 0, 14, 21, 28, 35 and Day 7 post boost. The average relative absorbance density read at 450 821

nm from three individual samples were plotted graphically. (B) HI titers were measured from 822

ferret sera against inactivated 2007-2008 seasonal Solomon Island A/Solomon Islands/3 H1N1, 823

A/Wisconsin/67/2005 H3N2 and B/Malaysia/2507/2004 viruses. (C) Neutralizing antibody titers 824

for blocking the live A/Solomon Islands/3 H1N1 virus were measure by microneutralization 825

assays. Three independent experiments were performed. Error bars indicate standard deviation. 826

Statistical analysis between adjuvant added vaccine and vaccine alone treated group animals 827

were performed. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001. 828

Figure 2. CpG ODN vaccine activated ISGs expression early post immunization. 829

mRNA expression profiles from immunized ferrets with and without CpG ODN were determined 830

by Q-PCR. (A) The heat map represents the mRNA expression profile of 50 ferret immune-831

related genes in V2007

+ CpG (V + CpG) and V2007

-Alone (V-Alone) group animals (n = 3/group) 832

at Day 1 post vaccination. The heat map was generated by Multiexperiment Viewer software 833

version 4.1 from the β-actin normalized real-time PCR data relative to PBS control group. Genes 834

were listed by descending mRNA-level (red, upregulation; green, downregulation). V represents 835

the V2007

. (B) Transcription of nine ISG genes (OAS1, RIG-I, Mx-1, CXCL10/IP10, ISG15, 836

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IRF1, IRF7, STAT1 and STAT2) in ferret whole blood was quantified by Q-PCR and displayed 837

graphically. The PBS V2007

boost group animals were treated with PBS at Day 0 and boosted 838

with commercial vaccine at Day 35 after injection. The RNA samples used for expression 839

analysis were extracted from the peripheral blood of three ferrets (n = 3) in each group at every 840

time point. mRNA levels were normalized to β-actin and then to PBS control groups. The data 841

was averaged from three independent experiments and the error bars represent standard 842

deviation. Horizontal bars indicate the statistical analysis performed between the selected two 843

groups. *: p ≤ 0.05, **: p ≤ 0.01. 844

Figure 3. The levels of ISG regulatory genes in CpG ODN adjuvant-mediated 845

immunization. 846

The average transcription levels of (A) IFN-α and IFN-γ and (B) IRF4 in CpG ODN mediated 847

immunization were determined by Q-PCR and plotted graphically from various time points 848

following immunization. Increases in mRNA levels were relative to β-actin and then normalized 849

to PBS control groups. Average was obtained from three independent experiments and the error 850

bars indicate standard deviation. Horizontal bars show the statistical analysis performed between 851

the selected two groups. *: p ≤ 0.05. 852

Figure 4. IFN-α and CFA-mediated vaccination induced influenza virus-specific antibody 853

production. 854

(A) Antibody titers in vaccinated ferret serum against 2008-2009 seasonal live 855

A/Brisbane/59/2007 H1N1 virus and A/Brisbane/10/2007 H3N2 virus were measured by HI 856

inhibition assays. (B) Neutralizing antibody titers for blocking the live A/Brisbane/59/2007 857

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H1N1 virus were measured from immunized ferrets by microneutralization assays. Data 858

represents triplicate measurements of sera collected from three animals. Error bars indicate 859

standard deviation. Statistical analysis between adjuvant added vaccine and vaccine alone treated 860

group animals were performed by either one-way ANOVA in figure (A) or student t test in figure 861

(B). *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001. 862

Figure 5. Adjuvant-mediated vaccination decreased viral load following influenza 863

infection. 864

Viral load in nasal wash at Day 3 post H1N1 influenza challenge was determined. Nasal wash 865

from each group animals (n = 3) was collected post infection, cultured and titrated. The virus 866

titer was calculated by Reed-Muench method and expressed as TCID50/ml. Error bars 867

demonstrate standard error of the mean. Statistical analysis was performed between each 868

adjuvant added vaccination group and PBS control group by student t test. V represents the 869

V2008

. *: p ≤ 0.05, **: p ≤ 0.01. 870

Figure 6. Memory antibody response at Day 5 post H1N1 infection was increased in 871

adjuvant-mediated vaccinated animals. 872

(A) Antibody titers from ferret sera against live seasonal A/Brisbane/59/2007 H1N1 virus was 873

determined by HA inhibition assays at Day 5 post infection for adjuvant and non-adjuvant 874

assisted vaccinations. (B) Neutralizing antibody titers for blocking the A/Brisbane/59/2007 875

H1N1 virus were assessed by microneutralization assays. The statistical analysis determined 876

between Day 35 after vaccination and Day 5 post infection is shown by horizontal bar. The 877

differences between adjuvant groups and vaccine alone group at Day 5 p.i. were analyzed by 878

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one-way ANOVA. Data represents the average of triplicate measurements of sera collected from 879

three animals. Error bars indicate standard deviation. V represents the V2008

. *: p ≤ 0.05, **: p ≤ 880

0.01, ***: p ≤ 0.001. 881

Figure 7. Adjuvant-mediated vaccination activated differing arms of innate and adaptive 882

immunity. 883

The heat maps represent RNA expression determined by microarray analysis Day 1 post 884

vaccination. The heat map generated from the gene expression profile of the four different 885

vaccination groups (n = 3/group) shows the innate, adaptive, Ag processing/presentation and 886

complement immune related genes, which were significantly regulated by at least 1.5 fold 887

change (p ≤ 0.05) in one of the adjuvant additive groups. The fold change of the gene expression 888

is shown in table 3 (red, upregulation; blue, downregulation). V represents the V2008

. 889

Figure 8. CpG ODN and PEG-IFN-adjuvanted vaccine-regulated IFN signaling pathways. 890

(A) IFN-responsive gene expression was determined by microarray analysis from the RNA of 891

vaccinated ferrets at Day 1 post vaccination (red, upregulation; blue, downregulation) and plotted 892

by heat map. (B) A schematic was created from the gene expression profiling data obtained Day 893

1 post immunization by IPA: the upper panel shows the IPA canonical pathway of IFN signaling 894

using the Q-PCR of 2007 vaccine study and microarray data of the 2008 vaccine study from 895

CpG-mediated immunized ferrets; the lower panel exhibits the IFN signaling pathway activated 896

by PEG-IFN-adjuvanted vaccination by using the microarray data of the 2008 vaccine study (red, 897

upregulation). (C) Gene expression heat map of molecules involved in Ras signaling at Day 1 898

post-vaccination (upper panel; red, upregulation), and IPA modeling of JAK/STAT pathway-899

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mediated Ras signaling using the microarray data from the whole blood RNA of PEG-IFN-900

mediated immunized ferrets at Day 1 post vaccination (lower panel; red, upregulation). 901

Figure 9. Cell adhesion and B cell signaling pathways were activated in ferrets immunized 902

with CFA-mediated vaccines. 903

(A) Heat map of cell adhesion molecule gene expression and genes involved in B cell 904

activation/calcineurin-NFAT signaling at Day 1 post vaccination (red, upregulation; blue, 905

downregulation). (B), (C) and (D) IPA canonical pathways of integrin signaling, B cell 906

activation, and calcineurin-NFAT created by using the microarray data of CFA immunized ferret 907

peripheral blood RNA Day 1 post vaccination, (red, upregulation; green, downregulation). V 908

represents the V2008

. 909

Figure S1. The level of ferret_DLA-64-like, ferret_FCN1-like and ferret_SOCS3-like 910

expression as quantified by Q-PCR in adjuvant-mediated vaccination. 911

To confirm the gene changes from the microarray analysis, the average transcription levels of 912

three selected genes (A) Ferret_DLA-64-like, (B) Ferret_FCN1-like and (C) Ferret_SOCS3-like 913

in multiple vaccination groups (n=3/group) were determined by Q-PCR at day 1 following 914

immunization. Increases in mRNA levels were relative to β-actin and then normalized to PBS 915

control groups. Average values were obtained from three independent experiments and the error 916

bars indicate standard deviation. The asterisk showed the statistical difference found between the 917

adjuvanted vaccine group and vaccine alone group. *: p ≤ 0.05. 918

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Table 1. Ferret Specific Gene Primer list

Primer NameATCC Item

Number

NCBI Gene

Accesion NumberPrimer 5'-3' Sequence

Caspase 4 F NR-8075 EU836041 TCATTGCTTTCTGCTCCTCA

Caspase 4 R NR-8076 EU836041 ATCTGGGCTTTCACATCTGG

C1 Inhibitor F NR-8079 EU835994 GCCTCTCAGAGCCTGTATGG

C1 Inhibitor R NR-8080 EU835994 CTTCCACTTGGCACTCAGGT

Hsp40 F NR-8081 EU836010 CCACCGTGTCGAGCTTTATT

Hsp40 R NR-8082 EU836010 CATGAACGTTGGACTGGTTG

NFAT5 F NR-8083 EU836021 ACCTCTTCCAGCCCTACCAT

NFAT5 R NR-8084 EU836021 CCTCTTCGGTGTTGATGGAT

SOCS5 F NR-8087 EU835993 GTTAGCTCCCGGAATGACTG

SOCS5 R NR-8088 EU835993 CTTTCCAAGCTCCCTGTCTG

IRF2 F NR-8091 EU835486 AGGTGACCACCGAGAGTGAC

IRF2 R NR-8092 EU835486 CCCCATGTTGCTGAGGTACT

CD3 F NR-8097 EF492054 GGCGGTGGCTGCAATC

CD3 R NR-8098 EF492054 TCCAGTAATAGACCAGCAGAAGCA

CD14 F NR-8103 TBA AGTGCCATCGAGGTGGAGAT

CD14 R NR-8104 TBA GCGTACTGCTTCGGGTCTGT

CRLF2 F NR-8109 EU836018 GGCTTGGATGCTGAGAAATGTT

CRLF2 R NR-8110 EU836018 GGGCCATAGCTGGACTCCAT

HLA-DRA_like F NR-8115 EU835995 GAGAGCCCAACATCCTCATCTG

HLA-DRA_like R NR-8116 EU835995 TCGAAGCCACGTGACATTGA

STAT3 F NR-8121 EU835482 CAACCCCAAGAACGTGAACT

STAT3 R NR-8122 EU835482 AGCCCACGTAATCTGACACC

TLR2 F NR-8123 EU836009 TCTTCTGGAGCCCATTGAGAA

TLR2 R NR-8124 EU836009 GTGTTCATTATCTTCCGCAGCTT

NFKBIA F NR-8125 EU835992 CCAGCACCTCTACTCCATCC

NFKBIA R NR-8126 EU835992 CATCAGCACCCAAAGACACC

Sprouty 4 F NR-8127 EU836020 CAGCGGCTCTTGGACCAC

Sprouty 4 R NR-8128 EU836020 ACTTACACTTCCCACAGG

IFN-alpha F NR-8133 EF492061 TCTCCATGTGACAAACCAGAAGA

IFN-alpha R NR-8134 EF492061 CAGAAAGTCCTGAGCACAATTCC

IFN-gamma F NR-8137 EF492064 TCAAAGTGATGAATGATCTCTCACC

IFN-gamma R NR-8138 EF492064 GCCGGGAAACACACTGTGAC

IFI35 F NR-8141 EU835487 GGGCTCCGGCTGAGTGA

IFI35 R NR-8142 EU835487 CCACCCCCATTTCTGGTCTT

P52rIPK_like F NR-8143 EU835488 TACGATGCCGAACTTCTG

P52rIPK_like R NR-8144 EU835488 TGCTCTCCTACAATTCTCC

Mx1 F NR-8147 EU835483 ACATCCTCAGGCAGGAGACA

Mx1 R NR-8148 EU835483 CAGGTCAGGCTTTGTCAAGA

IL-1beta F NR-8149 EU835490 GGACTGCAAATTCCAGGACATAA

IL-1beta R NR-8150 EU835490 TTGGTTCACACTAGTTCCGTTGA

IL-4 F NR-8151 EF492062 TCACCGGCACTTTCATCCA

IL-4 R NR-8152 EF492062 TTCTCGCTGTGAGGATGTTCA

IL-6 F NR-8153 EF492063 AGTGGCTGAAACACGTAACAATTC

IL-6 R NR-8154 EF492063 ATGGCCCTCAGGCTGAACT

IL-8 F NR-8155 EU477256 AAGCAGGAAAACTGCCAAGAGA

IL-8 R NR-8156 EU477256 GCCAGAAGAAACCTGACCAAAG

IL-16 F NR-8157 EU835491 CCCACTCGGACCTTCT

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IL-16 R NR-8158 EU835491 GAGAGGAGCCAAAGGTCTCAA

NKG7 F NR-8159 EU836023 GCGAGCGGTGGAACCA

NKG7 R NR-8160 EU836023 CAGGTAGAACGACCAGGAGAAGA

CXCL10/IP10 F NR-8161 EF492058 CTTTGAACCAAAGTGCTGTTCTTATC

CXCL10/IP10 R NR-8162 EF492058 AGCGTGTAGTTCTAGAGAGAGGTACTC

TNF-alpha F NR-8167 EF492065 CCAGATGGCCTCCAACTAATCA

TNF-alpha R NR-8168 EF492065 GGCTTGTCACTTGGAGTTCGA

CXCR3 F NR-8169 EF492060 TTTGACCGCTACCTGAGCAT

CXCR3 R NR-8170 EF492060 GCCGACAGGAAGATGAAGTC

Beta-actin F NR-8171 EU836011 TGACCGGATGCAGAAGGA

Beta-actin R NR-8172 EU836011 CCGATCCACACCGAGTACTT

CCL5 F NR-8173 EU835495 GCTGCTTTGCCTACATTTCC

CCL5 R NR-8174 EU835495 CCCATTTCTTCTGTGGGTTG

NF-kB F NR-8177 EU835494 AGGATCGATCAAAGCCTGAA

NF-kB R NR-8178 EU835494 CCCTCACCAGGTAACAGAGC

TLR3 F NR-8179 EU835984 GATGACCTCCCAGCAAACAT

TLR3 R NR-8180 EU835984 GCACAATTCTGGCTCCAGTT

MyD88 F NR-8181 EU836043 GCGTTTTGATGCCTTCATCT

MyD88 R NR-8182 EU836043 GGCAAGACATCACGATCAGA

TRAF6 F NR-8183 EU836040 AGATTGGCAACTTTGGGATG

TRAF6 R NR-8184 EU836040 ACAGTTTGTAGCCGGGTTTG

IRAK4 F NR-8185 EU836022 CCCCTGCAAGTCTTTTGCTA

IRAK4 R NR-8186 EU836022 GATGTCCTGTCTTTGCCACA

OAS1 F NR-8187 EU835484 TGAAGAGCCTCCTACGGCTA

OAS1 R NR-8188 EU835484 CCATCTCCCAGGCGTAGATA

TLR4 F NR-8191 EU835996 AGTGGGTCAAAGACCACAGG

TLR4 R NR-8192 EU835996 GACAGGGTGGCATTCCTAAA

IRF7 F NR-8195 EU835985 ACACTCTACCCCCGTGTCTG

IRF7 R NR-8196 EU835985 GTCAAGTCCAGAGCCTCCAA

ISG20 F NR-8197 TBA GGTGGCCATTGACTGTGAG

ISG20 R NR-8198 TBA GCCGGATGAACTTGTCGTAG

TLR9 F NR-8201 TBA ACTCCGACTTTGTCCACCTG

TLR9 R NR-8202 TBA GGTCATGTGACAGGGGAAGT

CASP8 F NR-9500 EU836044 ATTGCCAATGTCGGACTCTC

CASP8 R NR-9501 EU836044 TACTCAACGCATCTGCATCC

ICAM1 F NR-9508 EU836045 TGGACTACGGTGACTGTGGA

ICAM1 R NR-9509 EU836045 CGGACAATCCCTCTGGTCTA

IL8RB F NR-9512 EU836026 CGCTCCTGAAGGAAGTCAAC

IL8RB R NR-9513 EU836026 AGCAGACTGGGCTGGAGTAG

IRF1 F NR-9514 EU835485 CGATACAAAGCAGGGGAAAA

IRF1 R NR-9515 EU835485 GGCCTTGCACTTAGCATCTC

IRF4 F NR-9516 EU836039 AATCCTCGTGAAGGAGCTGA

IRF4 R NR-9517 EU836039 AGATCCTGCTCTGGCACAGT

ISG15 F NR-9518 EU835986 AGCAGCAGATAGCCCTGAAA

ISG15 R NR-9519 EU835986 CAGTTCTTCACCACCAGCAG

PKR F NR-9522 EU835989 ACGAATACGGCATGAAGACC

PKR R NR-9523 EU835989 TGGAAGGGTCAGGCATTAAG

RIG-I F NR-9524 EU836024 AGAGCACTTGTGGACGCTTT

RIG-I R NR-9525 EU836024 TGCAATGTCAATGCCTTCAT

SOCS3 F NR-9528 EU835987 GCTGGTGCATCACTACATGC

SOCS3 R NR-9529 EU835987 GACCGTCTTCCGACAGAGAT

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STAT2 F NR-9530 EU835988 AGCTGCTGAAGGAGCTGAAG

STAT2 R NR-9531 EU835988 TGCCTTCCTGGAGTCTCACT

STAT1 F not available EU835493 AGCCTTGCATGCCAACTCA

STAT1 R not available EU835493 ACAGTCCAGCTTCACCGTGAA

FCN1 F NR-8117 TBA CACCAAGGACCAGGACAATGA

FCN1 R NR-8118 TBA CACCAGGCCCCCTGGTA

DLA-64_like F not available TBA CAGGACACAGAGGTTGTGGA

DLA-64_like R not available TBA TGGCACGTGTATCTCTGCTC

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Table 2. Inactivity of Infected Ferrets

GROUP

Relative

Inactivity Index Day 1 Day 2 Day 3 Day 4 . Day 5

PBS 1.6 0.0 1.5 0.6 0.5 0.5

V + CFA 1.1 0.0 0.0 0.5 0.0 0.0

V + CpG 1.3 0.0 0.6 0.5 0.3 0.3

V + IFN 1.3 0.0 0.5 0.3 0.4 0.4

V + PBS 2.0 0.5 1.4 1.4 0.9 0.6

Relative inactivity index was calculated from daily activity scores as described in Materials and Methods

Average of daily activity score was obtained from 3 animals in each group

2008-2009 human flu vaccine

Daily Mean Inactivity Score Post Infection

a

b

a

b

C

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Table 3. In vivo differential microarray gene expression analysis on whole blood of ferrets immunized with adjuvanted 2008-2009 vaccines (n=3) versus PBS controls (n=3) on day 1 post vaccination.

Gene Symbol

V + CpG V + IFN V + CFA V + PBS

IFN Responding Genes

STAT1 2.7 2.3 1.1 1.0

IRF1 2.3 1.2 1.5 1.1

IRF2 1.5 2.5 2.3 -1.1

OAS1 3.2 2.3 -1.1 -1.0

OAS2 2.4 1.8 -1.1 -1.0

OASL 1.9 1.1 1.0 1.1

ISG15 7.6 6.9 1.4 -1.0

USP18 4.8 3.2 1.1 -1.0

IFI44 3.4 3.3 1.7 -1.1

IFI44L 3.4 3.3 1.7 -1.0

IFIT2 1.7 1.5 1.4 1.1

RSAD2 5.6 3.5 2.2 1.2

EIF2AK2 (PKR) 1.7 1.3 1.1 -1.1

CXCL10 1.6 1.1 1.0 1.3

SOCS1 2.7 1.2 1.0 1.3

Toll-like Receptor

TLR4 1.5 1.1 1.1 1.0

Antigen Processing and Presentation Gene

CTSB 1.6 1.4 1.1 -1.0

CTSS 1.7 1.4 1.2 -1.0

CTSD 1.1 1.9 1.7 -1.0

MHC class Ib 1.1 1.5 1.3 1.0

MHC class I DLA-64 1.1 1.4 1.9 1.1

HLA-DMA 1.1 1.4 2.0 -1.1

CD74 1.0 2.1 1.9 1.0

RFX5 1.3 1.5 1.6 1.0

CIITA 1.1 1.1 -1.8 -1.0

Adaptive Immunity Regulator

LYN 1.2 1.2 1.5 1.0

SYK 1.0 1.4 1.6 -1.1

PIK3CD 1.2 1.6 1.8 -1.1

RAC1 1.2 1.2 2.3 1.1

BAM32 (DAPP1) 1.3 1.1 1.5 -1.1

HRAS 1.3 2.0 2.5 1.0

RAF1 -1.1 1.5 1.8 1.0

MAP2K2 1.0 1.6 1.5 1.1

MAPK1 1.0 1.0 1.6 1.0

NFATC3 1.1 1.2 2.0 -1.2

AKT3 -1.1 -1.1 -1.7 -1.4

Mean of Fold Changea b

C

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POU2F2 1.1 1.3 1.6 -1.2

HoxC4 1.1 -1.1 -1.7 -1.1

AICDA (AID) 1.0 1.1 2.6 1.0

JUNB 1.3 2.3 2.8 1.2

RGS1 2.3 1.6 1.0 1.2

GNA15 1.3 2.0 2.1 1.2

GNG2 1.1 1.4 1.6 1.1

GNG11 1.0 1.2 1.6 1.1

GNB3 1.1 1.4 1.8 -1.1

Calcineurin (PPP3R1) 1.0 1.2 1.7 -1.0

RCAN1 1.1 -1.1 -2.3 1.0

CSNK1D 1.0 1.1 1.9 1.1

CSNK1G2 1.1 1.1 1.5 -1.1

MEF2C -1.1 -1.2 1.9 -1.2

LAT -1.1 1.1 -1.5 -1.0

IL12A -1.1 -1.4 -1.8 -1.1

SOCS3 1.2 1.2 1.6 -1.0

CXCL14 1.3 1.4 1.6 1.1

Complement

C3 precursor 2.4 2.4 1.1 -1.0

FCN1 2.6 1.9 1.1 -1.0

C1QL2 1.2 1.1 1.5 -1.0

C1R 1.1 1.5 3.9 1.1

C5AR1 1.3 1.3 1.6 -1.0

CFI 1.0 1.0 1.7 -1.0

Cell Adhesion Molecule and Regulator

VCAM1 -1.1 1.3 1.9 -1.1

BCAM 1.1 1.3 2.0 -1.0

CD36 1.1 1.2 1.6 1.0

CD44 1.1 1.1 1.5 1.0

CD84 1.4 1.6 1.7 1.2

Integrin-beta6 1.1 1.1 1.6 1.0

ZYXIN 1.2 1.7 2.6 1.2

TALIN 1 1.1 1.0 1.5 -1.1

RHOG 1.2 1.4 1.9 -1.1

RHOT1 1.0 -1.1 1.8 -1.1

CRKL 1.0 1.0 1.5 -1.1

RAP2A 1.0 1.2 1.9 1.1

The gene symbol is compatible to the human ortholog

The mean fold change is normalized to the corresponding PBS control group. Boldface indicates

the gene expression is significanltly induced by at least 1.5 fold change (p г 0.05) versus controls.

2008-2009 human flu vaccine

b

C

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Ferret Antigen Sepcific IgM

Days

Day0 Day14 Day21 Day28 Day35Day7 Post Boost

OD

45

0

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

**

PBS

V2007

-Alone

V2007

+CpG

H1N1 HA Inhibition Test

Days

Day0 Day14 Day21 Day28 Day35 Day7 Post Boost

HI

Titer

(Lo

g2)

4

6

8

10

12

** ** *****

**PBS

PBS V2007

Boost

V2007

-Alone

V2007

+CpG

Influenza B Strain HA Inhibition Test

Days

Day0 Day14 Day21 Day28 Day35 Day7 Post Boost

HI T

ite

r (L

og

2)

4

6

8

10

12

** * ***

**

Microneutralization Assay on Solomon Islands H1N1 Virus

Days

Day0 Day14 Day21 Day28 Day35 Day7 Post Boost

Ne

utr

aliz

ing

Antibo

dy T

ite

r (L

og

2)

8

10

12

14

16 PBS

PBS V2007

Boost

V2007

-Alone

V2007

+CpG

** **

***

**

Ferret Antigen Specific IgG

Days

Day0 Day14 Day21 Day28 Day35Day7 Post Boost

OD

45

00.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

**

****

**

Fig.1

A

H3N2 HA Inhibition Test

Days

Day0 Day14 Day21 Day28 Day35 Day7 Post Boost

HI

Titer

(Lo

g2)

4

6

8

10

12

*** ******

***

***

B

C

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Fig.2

RIG-I Q-PCR Assay

Days

Day1 Day3 Day5 Day7 Post Boost

Norm

aliz

ed to B

-actin

0

2

4

6

8

* **

* *

Mx-1 Q-PCR Assay

Days

Day1 Day3 Day5 Day7 Post Boost

Norm

aliz

ed to B

-actin

0

1

2

3

4

*

* *

CXCL10 Q-PCR Assay

Days

Day1 Day3 Day5 Day7 Post Boost

Norm

aliz

ed to B

-actin

0

1

2

3

4

5

6

7

*

**

ISG15 Q-PCR Assay

Days

Day1 Day3 Day5 Day7 Post Boost

Norm

aliz

ed t

o B

-actin

0

1

2

3

4

5

6

** **

IRF1 Q-PCR Assay

Days

Day1 Day3 Day5 Day7 Post Boost

No

rmaliz

ed

to B

-actin

0.0

0.5

1.0

1.5

2.0

2.5

*

IRF7 Q-PCR Assay

Days

Day1 Day3 Day5 Day7 Post Boost

Norm

aliz

ed to B

-actin

0.0

0.5

1.0

1.5

2.0

2.5

*** *

STAT1 Q-PCR Assay

Days

Day1 Day3 Day5 Day7 Post Boost

Norm

aliz

ed

to

B-a

ctin

0.0

0.5

1.0

1.5

2.0

2.5

** *

STAT2 Q-PCR Assay

Days

Day1 Day3 Day5 Day7 Post Boost

Norm

aliz

ed

to

B-a

ctin

0.0

0.5

1.0

1.5

2.0

2.5

3.0

* ***

A BOAS1 Q-PCR Assay

Days

Day1 Day3 Day5 Day7 Post Boost

No

rma

lize

d to

B-a

ctin

0

1

2

3

4

5

**

***

*PBS V

2007 Boost

V2007

-Alone

V2007

+CpG

V-Alone V+CpG

-3 30

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IFN-gamma Q-PCR Assay

Days

Day1 Day3 Day5 Day7 Post Boost

Norm

aliz

ed

to B

-actin

0.0

0.5

1.0

1.5

2.0

2.5

Fig.3IFN-alpha Q-PCR Assay

Days

Day1 Day3 Day5 Day7 Post Boost

Norm

aliz

ed

to

B-a

ctin

0

1

2

3

4

5

*

**PBS V

2007 Boost

V2007

-Alone

V2007

+CpG

IRF4 Q-PCR Assay

Days

Day1 Day3 Day5 Day7 Post Boost

Norm

aliz

ed

to

B-a

ctin

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

*

*

A

B

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Microneutraliztion Assay on Brisbane59 H1N1 Virus

Days

Day0 Day14 Day21 Day28 Day35

Neu

tralizin

g A

nti

bo

dy T

iter

(Lo

g2)

8

10

12

14

16

PBS

V2008

-Alone

V2008

+CpG

V2008

+IFN

V2008

+CFA

*

*

* **

***

Fig.4

A

B

H1N1 HA Inhibition Test

Days

Day0 Day14 Day21 Day28 Day35

HI

Tite

r (L

og

2)

4

6

8

10

12

PBS

V2008

-Alone

V2008

+CpG

V2008

+IFN

V2008

+CFA

***

***

******

H3N2 HA Inhibition Test

Days

Day0 Day14 Day21 Day28 Day35

HI T

iter

(Log

2)

4

6

8

10

12

** *** *** ***

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Day 5 Post Infection

Vaccination

PBS V-Alone V+CpG V+IFN V+CFA

HI T

iter

(Log2)

Blo

ckin

g H

1N

1 V

irus

3

4

5

6

7

8

9

***

Fig.5

Viral Load in Nasal Wash at Day 3 Post Infection

Vaccination

PBS V-Alone V+CpG V+IFN V+CFA

TC

ID5

0/m

l (L

og

10)

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

**

*

**

Fig.6

A BMemory Antibody Response to H1N1

Days

Day14 Day21 Day28 Day35 Day5 Post Infection

Ne

utr

aliz

ing

An

tib

od

y T

ite

r (L

og

2)

8

10

12

14

16PBS

V2008

-Alone

V2008

+CpG

V2008

+IFN

V2008

+CFA

**

*

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Fig.7

-3 30

Innate Immune Defense Gene

V-AloneV+CFAV+IFNV+CpG

ComplementV-AloneV+CFAV+IFNV+CpG

V-AloneV+CFAV+IFNV+CpG

Ag Processing and Presentation

V-AloneV+CFAV+IFNV+CpG

Adaptive Immune Defense Gene

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Fig.8

B

-3 30

Interferon Stimulated GeneA

V+CpG

V+IFN

V-AloneV+CFAV+IFNV+CpG

-3 30

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Fig.8

Ras Signaling MoleculeC

-3 30

V-AloneV+CFAV+IFNV+CpG

-3 30

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Fig.9

Cell Adhesion and Motility B cell ActivationA

B

-3 30

V-AloneV+CFAV+IFNV+CpGV-AloneV+CFAV+IFNV+CpG

-3 30

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Fig.9

C

D

-3 30

-3 30

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