A Randomised, Double-blind, Placebo-controlled Phase IIb Trial to Test FLU-v Vaccine

• ClinicalTrials.gov Identifier: NCT02962908
• Recruitment Status: Completed
• First Posted: November 15, 2016
• Results First Posted: April 8, 2019
• Last Update Posted: September 16, 2020
• Sponsor: PepTcell Limited
• Collaborators: Seventh Framework Programme; University of Groningen; University Medical Center Groningen; Robert Koch Institut; Norwegian Institute of Public Health
• Information provided by (Responsible Party): PepTcell Limited

• Study Type: Interventional
• Study Design: Allocation: Randomized; Intervention Model: Parallel Assignment; Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor); Primary Purpose: Prevention
• Condition: Influenza
• Interventions: Biological: FLU-v; Biological: adjuvanted FLU-v; Biological: Saline; Biological: Adjuvanted placebo
• Enrollment: 175

Participant Flow

Recruitment Details
Pre-assignment Details	195 subjects were screened. Of those, N=20 subjects were removed from the study prior randomisation due to: lost to follow up n=3; didn't wish to continue after screening visit n=9; did not meet inclusion/exclusion criteria n=8

Arm/Group Title	2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description	FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Period Title: Overall Study
Started	58	58	32	27
Completed	58	50	32	24
Not Completed	0	8	0	3
Reason Not Completed
Lost to Follow-up	0	3	0	1
Withdrawal by Subject	0	3	0	2
Protocol Violation	0	1	0	0
Physician Decision	0	1	0	0

Baseline Characteristics

Arm/Group Title		2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo	Total
Arm/Group Description		FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection	Total of all reporting groups
Overall Number of Baseline Participants		58	57	32	27	174
Baseline Analysis Population Description		A subject in 1x FLU-v adjuvanted group received 1x non-adjuvanted FLU-v. Subject completed the study but with this treatment, the subject could not be assigned to any group for analysis. This is the reason why 58 subjects were randomised to this group but only 57 were analysed, making the total subject number 174 rather than 175.
Age, Continuous; Mean (Standard Deviation); Unit of measure: Years
	Number Analyzed	58 participants	57 participants	32 participants	27 participants	174 participants
		40.02 (13.691)	40.12 (12.221)	41.19 (12.458)	39.07 (13.074)	40.12 (12.806)
Sex: Female, Male; Measure Type: Count of Participants; Unit of measure: Participants
	Number Analyzed	58 participants	57 participants	32 participants	27 participants	174 participants
	Female	36 62.1%	30 52.6%	18 56.3%	13 48.1%	97 55.7%
	Male	22 37.9%	27 47.4%	14 43.8%	14 51.9%	77 44.3%
Race/Ethnicity, Customized; Measure Type: Count of Participants; Unit of measure: Participants	Number Analyzed	58 participants	57 participants	32 participants	27 participants	174 participants
Asian		1 1.7%	1 1.8%	0 0.0%	0 0.0%	2 1.1%
Black of African descendant		0 0.0%	0 0.0%	0 0.0%	0 0.0%	0 0.0%
White		56 96.6%	54 94.7%	32 100.0%	27 100.0%	169 97.1%
Other		1 1.7%	2 3.5%	0 0.0%	0 0.0%	3 1.7%
Unknown or not reported		0 0.0%	0 0.0%	0 0.0%	0 0.0%	0 0.0%
previous influenza vaccination [1] [2]; Measure Type: Count of Participants; Unit of measure: Participants
	Number Analyzed	58 participants	57 participants	32 participants	27 participants	174 participants
	Never received influenza vaccination	37 63.8%	29 50.9%	15 46.9%	18 66.7%	99 56.9%
	Received in the previous 2 years	14 24.1%	20 35.1%	12 37.5%	5 18.5%	51 29.3%
	Received over 2 years ago	7 12.1%	8 14.0%	5 15.6%	4 14.8%	24 13.8%
		[1] Measure Description: Subjects were asked whether they had received previous influenza vaccinations and whether it was within 2 years or more from joining the trial.; [2] Measure Analysis Population Description: This baseline measure was only analysed in the FAS population, that is the Full Analysis Set which includes all those subjects that completed vaccination and had at least pre-vaccination immunogenicity data and at least one post-vaccination time point.
HAI titer at screening [1]; Measure Type: Count of Participants; Unit of measure: Participants	Number Analyzed	58 participants	57 participants	32 participants	27 participants	174 participants
HAI≥80 B/Phuket/3073/2013		21 36.2%	16 28.1%	12 37.5%	7 25.9%	56 32.2%
HAI≥80 B/Brisbane/60/2008		18 31.0%	19 33.3%	12 37.5%	7 25.9%	56 32.2%
HAI≥40 A/Hong Kong/5738/2014 (H3N2)		25 43.1%	26 45.6%	18 56.3%	10 37.0%	79 45.4%
HAI≥40 A/Michigan/45/15 (H1N1)		23 39.7%	29 50.9%	16 50.0%	11 40.7%	79 45.4%
any of the above		39 67.2%	40 70.2%	26 81.3%	17 63.0%	122 70.1%
		[1] Measure Description: Serum samples were collected at screening (summer-autumn 2016) and later analysed for pre-existing neutralising antibodies against the main influenza strains that circulated in the Netherlands during the influenza season of 2016-2017 (B/Phuket/3073/2013, B/Brisbane/60/2008, A/Hong Kong/5738/2014 (H3N2) and A/Michigan/45/15 (H1N1)). The number of subjects with a HA titer for A strains ≥ 40 and with HA ≥ 80 for B strains was recorded in each group.

Outcome Measures

1. Primary Outcome

Title	CD4+ and CD8+ Th1 Cellular Immunogenicity on Day 42
Description	Comparison of the TH1 response in the treatment arms compared to placebo from baseline to day 42 following vaccination, calculated as the median fold change in the number of CD4+ and CD8+ T cells positive for IFN-gamma, TNF-alpha, IL-2 and CD107a. Fold change is calculated as the number of cells on day 42 divided by number of cells on day 0.
Time Frame	day 0 to day 42

Outcome Measure Data

Analysis Population Description

FAS population: Full Analysis Set corresponds to subjects that completed the vaccination successfully and provided samples for immunogenicity analysis for baseline (pre-vaccination) and at least one time point post-vaccination. Only samples with acceptable positive and negative controls were used in the analysis.

Arm/Group Title	2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description:	FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Overall Number of Participants Analyzed	58	51	32	26
Median (95% Confidence Interval); Unit of Measure: fold change
IFN-gamma CD4+	0.5; (-0.2 to 1.1)	5.9; (1.0 to 10.9)	1.0; (-0.7 to 2.6)	0.2; (-0.6 to 1.0)
TNF-alpha CD4+	0.0; (-0.1 to 0.1)	5.0; (-10.9 to 20.9)	0.0; (-0.5 to 0.5)	0.0; (-0.3 to 0.3)
IL-2 CD4+	0.0; (-1.1 to 1.1)	8.2; (-0.4 to 16.7)	0.0; (-0.4 to 0.4)	0.0; (-0.1 to 0.1)
CD107a CD4+	0.0; (-0.4 to 0.4)	2.0; (0.3 to 3.7)	0.0; (-0.5 to 0.5)	0.0; (-0.2 to 0.2)
IFN-gamma CD8+	0.3; (-0.1 to 0.6)	0.3; (-0.2 to 0.7)	0.0; (-0.1 to 0.1)	0.0; (-0.3 to 0.3)
TNF-alpha CD8+	0.3; (0.0 to 0.6)	0.3; (-0.6 to 1.3)	0.0; (-0.6 to 0.6)	0.0; (-0.1 to 0.1)
IL-2 CD8+	0.5; (0.1 to 0.9)	0.0; (-0.4 to 0.4)	0.0; (-0.5 to 0.5)	0.0; (-0.7 to 0.7)
CD107a CD8+	0.0; (-0.2 to 0.2)	0.0; (-0.5 to 0.5)	0.0; (-0.2 to 0.2)	0.0; (-0.1 to 0.1)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for IFN-gamma from day 0 and to 42.
	Type of Statistical Test	Other
	Comments	Inequality test.
Statistical Test of Hypothesis	P-Value	0.45
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 2

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for IFN-gamma from day 0 and to 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 3

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for TNF-alpha from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.88
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 4

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for TNF-alpha from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 5

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for IL-2 from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.21
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 6

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for IL-2 from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 7

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for CD107a from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.77
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 8

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for CD107a from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.004
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 9

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for IFN-gamma from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.08
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 10

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for IFN-gamma from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.38
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 11

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for TNF-alpha from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.49
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 12

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for TNF-alpha from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.156
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 13

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for IL-2 from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.125
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 14

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	[Not Specified]
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.89
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 15

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for CD107a from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.72
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 16

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for CD107a from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.34
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

2. Primary Outcome

Title	CD4+ and CD8+ Th1 Cellular Immunogenicity on Day 180
Description	Comparison of the TH1 response in the treatment arms compared to placebo from baseline to day 180 following vaccination, calculated as the median fold change in the number of CD4+ and CD8+ T cells positive for IFN-gamma, TNF-alpha, IL-2 and CD107a. Fold change is calculated as the number of cells on day 180 divided by number of cells on day 0.
Time Frame	Day 0 to day 180

Outcome Measure Data

Analysis Population Description

FAS population: Full Analysis Set corresponds to subjects that completed the vaccination successfully and provided samples for immunogenicity analysis for baseline (pre-vaccination) and at least one time point post-vaccination. Only samples with acceptable positive and negative controls were used in the analysis.

Arm/Group Title	2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description:	FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Overall Number of Participants Analyzed	58	51	32	26
Median (95% Confidence Interval); Unit of Measure: fold change
IFN-gamma CD4+	0.5; (0.2 to 1.2)	4.4; (1.0 to 7.8)	0.1; (-0.4 to 0.6)	0.9; (-1.4 to 3.2)
TNF-alpha CD4+	0.0; (0.0 to 0.0)	0.6; (-3.5 to 4.8)	0.0; (0.0 to 0.0)	0.0; (-1.2 to 1.2)
IL-2 CD4+	0.2; (-0.6 to 1.1)	6.5; (2.1 to 10.9)	0.0; (-0.2 to 0.2)	0.0; (-0.1 to 0.1)
CD107a CD4+	0.6; (-0.1 to 1.3)	1.0; (0.2 to 1.8)	0.0; (-0.1 to 0.1)	0.6; (-0.7 to 1.9)
IFN-gamma CD8+	0.0; (-0.5 to 0.6)	0.2; (-0.4 to 0.7)	0.0; (-0.6 to 0.6)	0.0; (-0.5 to 0.5)
TNF-alpha CD8+	0.1; (-0.5 to 0.6)	0.2; (-0.2 to 0.6)	0.1; (-0.7 to 0.9)	0.1; (-1.8 to 1.85)
IL-2 CD8+	0.3; (-0.3 to 0.7)	0.0; (-0.6 to 0.6)	0.0; (-0.3 to 0.3)	0.0; (-0.4 to 0.4)
CD107a CD8+	0.0; (-0.4 to 0.4)	0.0; (-0.9 to 1.5)	0.0; (-0.5 to 0.5)	0.5; (-3.2 to 6.8)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for IFN-gamma from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.62
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 2

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for IFN-gamma from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.030
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 3

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for TNF-alpha from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.87
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 4

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for TNF-alpha from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.075
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 5

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for IL-2 from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.076
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 6

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for IL-2 from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 7

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for CD107a from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.91
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 8

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD4+ T cells positive for CD107a from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.91
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 9

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for IFN-gamma from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.55
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 10

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for IFN-gamma from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.55
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 11

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for TNF-alpha from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality
Statistical Test of Hypothesis	P-Value	0.91
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 12

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for TNF-alpha from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.91
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 13

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for IL2 from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.21
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 14

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for IL2 from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.48
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 15

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for CD107a from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.62
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 16

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the difference in median fold increase in number of CD8+ T cells positive for CD107a from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	inequality
Statistical Test of Hypothesis	P-Value	0.23
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

3. Primary Outcome

Title	Percentage of TH1 Cytokine Responders (Responders Defined as Those With >2 Fold Median Increase From Baseline in CD4+ and CD8+ T-cells Positive for Particular Cytokine)
Description	To compare the number of subjects that showed at least a two-fold increase on day 42 and day 180 following vaccination in the number of CD4+and CD8+ T-cells secreting TH1 cytokines in all groups.
Time Frame	prevaccination, day 42 (21 days after last vaccination) and day 180.

Outcome Measure Data

Analysis Population Description

FAS population: Full Analysis Set corresponds to subjects that completed the vaccination successfully and provided samples for immunogenicity analysis for baseline (pre-vaccination) and at least one time point post-vaccination. Only samples with acceptable positive and negative controls were used in the analysis.

Arm/Group Title		2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description:		FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Overall Number of Participants Analyzed		58	51	32	26
Measure Type: Count of Participants; Unit of Measure: Participants
IFNg+CD4+ day 42	Number Analyzed	51 participants	44 participants	27 participants	19 participants
		23 45.1%	40 90.9%	13 48.1%	8 42.1%
TNFalpha+ CD4+ day 42	Number Analyzed	52 participants	44 participants	27 participants	18 participants
		16 30.8%	33 75.0%	12 44.4%	9 50.0%
IL2+ CD4+ day 42	Number Analyzed	52 participants	44 participants	27 participants	19 participants
		23 44.2%	36 81.8%	13 48.1%	4 21.1%
CD107a+ CD4+ day 42	Number Analyzed	47 participants	38 participants	23 participants	14 participants
		22 46.8%	26 68.4%	8 34.8%	4 28.6%
IFNg+ CD4+ day 180	Number Analyzed	53 participants	44 participants	27 participants	19 participants
		26 49.1%	37 84.1%	8 29.6%	11 57.9%
TNFa+ CD4+ day 180	Number Analyzed	53 participants	44 participants	27 participants	19 participants
		17 32.1%	26 59.1%	9 33.3%	9 47.4%
IL-2+ CD4+ day 180	Number Analyzed	53 participants	44 participants	27 participants	19 participants
		26 49.1%	39 88.6%	13 48.1%	9 47.4%
CD107a+ CD4+ day 180	Number Analyzed	50 participants	37 participants	22 participants	14 participants
		24 48.0%	21 56.8%	5 22.7%	8 57.1%
IFN-gamma day 42 CD8+	Number Analyzed	53 participants	44 participants	27 participants	18 participants
		22 41.5%	16 36.4%	9 33.3%	4 22.2%
TNF-alpha day 42 CD8+	Number Analyzed	53 participants	44 participants	27 participants	18 participants
		22 41.5%	23 52.3%	10 37.0%	6 33.3%
IL2 day 42 CD8+	Number Analyzed	52 participants	44 participants	27 participants	19 participants
		25 48.1%	17 38.6%	9 33.3%	8 42.1%
CD107a day 42 CD8+	Number Analyzed	47 participants	44 participants	27 participants	18 participants
		23 48.9%	19 43.2%	5 18.5%	2 11.1%
IFN-gamma day 180 CD8+	Number Analyzed	53 participants	44 participants	27 participants	19 participants
		27 50.9%	22 50.0%	13 48.1%	7 36.8%
TNF-alpha day 180 CD8+	Number Analyzed	53 participants	44 participants	27 participants	19 participants
		21 39.6%	18 40.9%	14 51.9%	12 63.2%
IL2 day 180 CD8+	Number Analyzed	53 participants	44 participants	27 participants	19 participants
		11 20.8%	16 36.4%	8 29.6%	5 26.3%
CD107a day 180 CD8+	Number Analyzed	53 participants	44 participants	27 participants	19 participants
		19 35.8%	24 54.5%	8 29.6%	8 42.1%

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD4+ IFNgamma responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.80
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 2

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD4+ IFNgamma responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 3

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD4+ TNF alpha responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.23
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 4

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD4+ TNF alpha responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.056
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 5

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD4+ IL-2 responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.74
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 6

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD4+ IL-2 responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 7

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD4+ CD107a responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.34
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 8

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD4+ CD107a responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.010
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 9

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD4+ IFNgamma responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.096
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 10

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD4+ IFNgamma responders on day 180.Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.049
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 11

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD4+ TNF alpha responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.91
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 12

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD4+ TNF alpha responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.39
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 13

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD4+ IL-2 responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.94
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 14

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD4+ IL-2 responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 15

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD4+ CD107a responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.044
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 16

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD4+ CD107a responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.98
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 17

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD8+ IFN-gamma responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.48
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 18

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD8+ IFN-gamma responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.28
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 19

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD8+ TNF-alpha responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.70
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 20

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD8+ TNF-alpha responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.175
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 21

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD8+ IL2 responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.24
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 22

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD8+ IL2 responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.80
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 23

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD8+ CD107a responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.023
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 24

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD8+ CD107a responders on day 42. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.015
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 25

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD8+ IFN-gamma responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.81
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 26

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD8+ IFN-gamma responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.34
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 27

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD8+ TNF-alpha responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.30
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 28

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD8+ TNF-alpha responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.105
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 29

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD8+ IL2 responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.38
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 30

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD8+ IL2 responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.44
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 31

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of CD8+ CD107a responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.58
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

Statistical Analysis 32

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of CD8+ CD107a responders on day 180. Differences considered significant if p-value <0.05.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.43
	Comments	[Not Specified]
	Method	Chi squared or Fisher's exact test
	Comments	[Not Specified]

4. Primary Outcome

Title	Percentage of CD4+ and CD8+ TH1 Cytokine Responders Determined by Mixture Models for Single-cell Assays (MIMOSA)
Description	To compare the number of subjects identified as responders for cytokine markers as determined by MIMOSA analysis (Responders based on a false discovery rate derived P-value <0.05).
Time Frame	day 0 to day 42 and day 180

Outcome Measure Data

Analysis Population Description

FAS: Full Analysis Set corresponds to subjects that completed the vaccination successfully and provided samples for immunogenicity analysis for baseline (pre-vaccination) and at least one time point post-vaccination. Only samples with acceptable positive and negative controls were used in the analysis.

Arm/Group Title		2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description:		FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Overall Number of Participants Analyzed		58	51	32	26
Measure Type: Count of Participants; Unit of Measure: Participants
IFN-gamma day 42 CD4+	Number Analyzed	54 participants	50 participants	30 participants	23 participants
		7 13.0%	38 76.0%	1 3.3%	2 8.7%
TNF-alpha day 42 CD4+	Number Analyzed	55 participants	50 participants	30 participants	22 participants
		3 5.5%	22 44.0%	1 3.3%	0 0.0%
IL-2 day 42 CD4+	Number Analyzed	55 participants	50 participants	30 participants	23 participants
		1 1.8%	28 56.0%	1 3.3%	0 0.0%
CD107a day 42 CD4+	Number Analyzed	51 participants	48 participants	29 participants	21 participants
		1 2.0%	5 10.4%	1 3.4%	0 0.0%
IFN-gamma day 180 CD4+	Number Analyzed	56 participants	49 participants	30 participants	21 participants
		8 14.3%	31 63.3%	2 6.7%	1 4.8%
TNF-alpha day 180 CD4+	Number Analyzed	56 participants	49 participants	30 participants	21 participants
		2 3.6%	12 24.5%	1 3.3%	0 0.0%
IL-2 day 180 CD4+	Number Analyzed	56 participants	49 participants	30 participants	21 participants
		1 1.8%	28 57.1%	1 3.3%	0 0.0%
CD107a day 180 CD4+	Number Analyzed	53 participants	44 participants	25 participants	19 participants
		2 3.8%	4 9.1%	2 8.0%	1 5.3%
IFN-gamma day 42 CD8+	Number Analyzed	56 participants	50 participants	30 participants	22 participants
		3 5.4%	3 6.0%	0 0.0%	0 0.0%
TNF-alpha day 42 CD8+	Number Analyzed	56 participants	50 participants	30 participants	22 participants
		0 0.0%	1 2.0%	0 0.0%	0 0.0%
IL2 day 42 CD8+	Number Analyzed	56 participants	50 participants	30 participants	23 participants
		0 0.0%	0 0.0%	0 0.0%	0 0.0%
CD107a day 42 CD8+	Number Analyzed	55 participants	50 participants	30 participants	22 participants
		0 0.0%	0 0.0%	0 0.0%	0 0.0%
IFN-gamma day 180 CD8+	Number Analyzed	56 participants	49 participants	30 participants	21 participants
		4 7.1%	3 6.1%	0 0.0%	1 4.8%
TNF-alpha day 180 CD8+	Number Analyzed	56 participants	49 participants	30 participants	21 participants
		1 1.8%	1 2.0%	0 0.0%	0 0.0%
IL2 day 180 CD8+	Number Analyzed	56 participants	49 participants	30 participants	21 participants
		0 0.0%	0 0.0%	0 0.0%	0 0.0%
CD107a day 180 CD8+	Number Analyzed	56 participants	49 participants	30 participants	21 participants
		0 0.0%	1 2.0%	0 0.0%	0 0.0%

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing IFN-gamma at day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.25
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 2

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing TNF-alpha at day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	1.0
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 3

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing IL-2 at day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	1.00
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 4

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing CD107a at day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	1.00
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 5

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing IFN-gamma at day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 6

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing TNF-alpha at day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 7

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing IL2 at day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 8

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing CD107a at day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.31
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 9

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing IFN-gamma at day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.48
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 10

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing TNF-alpha at day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	1.00
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 11

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing IL2 at day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	1.00
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 12

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing CD107a at day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.59
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 13

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing IFN-gamma at day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 14

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing TNF-alpha at day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.013
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 15

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing IL2 at day 180
	Type of Statistical Test	Other
	Comments	inequality
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 16

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD4+ T-cell producing CD107a at day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	1.00
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 17

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD8+ T-cell producing IFN-gamma at day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.55
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 18

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD8+ T-cell producing IFN-gamma at day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.55
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 19

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD8+ T-cell producing TNF-alpha at day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	1.00
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 20

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD8+ T-cell producing IFN-gamma at day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.29
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 21

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD8+ T-cell producing TNF-alpha at day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	1.00
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 22

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD8+ T-cell producing IFN-gamma at day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	1.00
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 23

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD8+ T-cell producing TNF-alpha at day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	1.00
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 24

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of responders determined by MIMOSA analysis of CD8+ T-cell producing CD107a at day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	1.00
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

5. Primary Outcome

Title	IFN-gamma Responses Measured by ELISA
Description	Median fold change in IFN-gamma secretion from PBMCs stimulated in vitro with FLU-v antigens. IFN-gamma secretion was measured by ELISA.Fold change was measured as secretion on day 42 divided by secretion on day 0, and secretion on day 180 divided by secretion on day 0.
Time Frame	day 0 to day 42, day 0 to day 180

Outcome Measure Data

Analysis Population Description

FAS: Full Analysis Set corresponds to subjects that completed the vaccination successfully and provided samples for immunogenicity analysis for baseline (pre-vaccination) and at least one time point post-vaccination. Only samples with acceptable positive and negative controls were used in the analysis.

Arm/Group Title		2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description:		FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Overall Number of Participants Analyzed		58	51	32	26
Median (95% Confidence Interval); Unit of Measure: fold change
Day 42	Number Analyzed	47 participants	43 participants	25 participants	20 participants
		0.8; (-2.3 to 3.9)	59.0; (33.3 to 84.7)	1.0; (-0.8 to 2.8)	1.0; (-2.4 to 4.4)
Day 180	Number Analyzed	48 participants	43 participants	23 participants	20 participants
		1.0; (0.7 to 1.3)	27.3; (5.2 to 49.4)	1.0; (-1.4 to 3.4)	0.9; (-0.0 to 1.9)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of median fold-increase in IFN-gamma secretion as measured by ELISA on day 42. p<0.05 considered statistically significant.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.59
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 2

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of median fold-increase in IFN-gamma secretion as measured by ELISA on day 42. p<0.05 considered statistically significant.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.001
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 3

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of median fold-increase in IFN-gamma secretion as measured by ELISA on day 180. p<0.05 considered statistically significant.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.61
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 4

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of median fold-increase in IFN-gamma secretion as measured by ELISA on day 180. p<0.05 considered statistically significant.
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

6. Primary Outcome

Title	Percentage of Responders on Day 42 and Day 180 for IFNgamma Secretion by PBMCs
Description	Responders were defined as subjects having at least a two-fold increase in the amount of IFNg secreted on day 42 and day 180 compared the amount secreted on day 0. IFNg was measured by ELISA
Time Frame	prevaccination (day 0) to postvaccination (day 42 and day 180)

Outcome Measure Data

Analysis Population Description

FAS: Full Analysis Set including subjects that completed vaccination and provided immunogenicity samples for prevaccination and at least one post-vaccination time point (day 42 or day 180). Only samples that meet the acceptance criteria based on the positive and negative controls were used in the analysis.

Arm/Group Title		2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description:		FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Overall Number of Participants Analyzed		58	51	32	26
Measure Type: Count of Participants; Unit of Measure: Participants
day 42	Number Analyzed	47 participants	44 participants	25 participants	20 participants
		28 59.6%	42 95.5%	10 40.0%	9 45.0%
day 180	Number Analyzed	48 participants	43 participants	23 participants	20 participants
		22 45.8%	40 93.0%	13 56.5%	11 55.0%

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of number of responders on day 42. A subject was considered a "responder" if an increase of secreted IFNgamma of at least two fold was observed from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.113
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 2

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of number of responders on day 42. A subject was considered a "responder" if an increase of secreted IFNgamma of at least two fold was observed from day 0 to day 42.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 3

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of number of responders on day 180. A subject was considered a "responder" if an increase of secreted IFNgamma of at least two fold was observed from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.399
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 4

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of number of responders on day 180. A subject was considered a "responder" if an increase of secreted IFNgamma of at least two fold was observed from day 0 to day 180.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

7. Primary Outcome

Title	Solicited AEs
Description	To evaluate the solicited AEs in all subjects
Time Frame	until 21 days after the last dosing of the study vaccine

Outcome Measure Data

Analysis Population Description

Safety Population: All subjects that received at least one vaccination

Arm/Group Title	2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description:	FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Overall Number of Participants Analyzed	58	57	32	27
Measure Type: Number; Unit of Measure: Events
All solicited AEs	275	485	149	147
Solicited AEs unrelated to vaccination	12	13	8	0
Solicited AEs unlikely related to vaccination	72	74	71	34
Solicited AEs possibly related to vaccination	108	134	64	39
Solicited AEs probably related to vaccination	16	16	0	12
Solicited AEs definately related to vaccination	67	248	6	62
Mild solicited AEs	210	339	124	111
Moderate solicited AEs	59	132	24	34
Severe solicited AEs	6	14	1	2

8. Secondary Outcome

Title	Antibody Responses to FLU-v
Description	To evaluate the IgG levels as a measure of antibody responses to FLU-v on day 0 (baseline) and on days 42 and 180 following FLU-v vaccination. IgG antibodies were measured by ELISA. The geometric mean for each treatment group was provided.
Time Frame	prevaccination, day 42 (21 days after last vaccination) and day 180.

Outcome Measure Data

Analysis Population Description

FAS: Full Analysis Set includes all subjects that completed the vaccination successfully and provided samples for immunogenicity analysis at baseline (Pre-vaccination) and at least one time point post-vaccination.

Arm/Group Title	2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description:	FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Overall Number of Participants Analyzed	58	51	32	26
Geometric Mean (Standard Error); Unit of Measure: IgG ng/ml
day 0	499.11 (103.19)	362.86 (82.93)	331.16 (59.00)	371.89 (67.47)
day 42	2593.02 (1652.34)	8740.48 (2432.90)	336.37 (58.92)	381.17 (61.18)
day 180	1276.34 (344.52)	4769.16 (1131.67)	344.88 (66.57)	387.26 (61.48)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of geometric mean IgG titers specific to FLU-v antigens on day 42 post-vaccination.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 2

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of geometric mean IgG titers specific to FLU-v antigens on day 42.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 3

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of geometric mean IgG titers specific to FLU-v antigens on day 180.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 4

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of geometric mean IgG titers specific to FLU-v antigens on day 180.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

9. Secondary Outcome

Title	Th2 Cytokine Responses (IL-4)
Description	To evaluate the level of TH2 cytokines (IL-4) from baseline in all groups 42 and 180 days following FLU-v vaccination.
Time Frame	prevaccination, day 42 (21 days after last vaccination) and day 180.

Outcome Measure Data

Analysis Population Description

FAS: Full Analysis Set includes all subjects that completed the vaccination successfully and provided samples for immunogenicity analysis at baseline (Pre-vaccination) and at least one time point post-vaccination. Only samples that met the acceptance criteria based on positive and negative controls were used in the analysis

Arm/Group Title	2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description:	FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Overall Number of Participants Analyzed	58	51	32	26
Measure Type: Number; Unit of Measure: percentage of responders
IL-4+CD4+ cells day 42	NA [1]	NA [1]	NA [1]	NA [1]
IL-4+ CD8+ cells day 42	NA [1]	NA [1]	NA [1]	NA [1]
IL-4+ CD4+ cells day 180	NA [1]	NA [1]	NA [1]	NA [1]
IL-4+ CD8+ cells day 180	NA [1]	NA [1]	NA [1]	NA [1]

[1]

Below level of detection

10. Other Pre-specified Outcome

Title	Unsolicited AEs and SAEs
Description	To evaluate unsolicited AEs and SAEs in all subjects
Time Frame	From the start of the vacciantion until study completion for each subject, approximately no more than 7 months

Outcome Measure Data

Analysis Population Description

Safety Population: all subjects that received at least one vaccination

Arm/Group Title	2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description:	FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Overall Number of Participants Analyzed	58	57	32	27
Measure Type: Number; Unit of Measure: events
All unsolicited AEs	19	24	13	9
Unsolicited AEs unrelated to the vaccine	8	11	5	5
Unsolicited AEs unlikely related to the vacccine	4	8	7	3
Unsolicited AEs possibly related to the vacccine	5	4	1	1
Unsolicited AEs probably related to the vaccine	0	0	0	0
Unsolicited AEs definately related to the vaccine	2	1	0	0
Mild unsolicited AEs	15	16	10	4
Moderate unsolicited AEs	3	4	1	5
Severe unsolicited AEs	1	3	2	0
Severe Adverse Events	2	3	0	0

11. Other Pre-specified Outcome

Title	Percentage of Participants Who Tested Positive for Influenza Strains
Description	During the influenza season (Dec 2016 to March 2017), fully vaccinated subjects will contact the trial center immediately if they feel unwell for 24h, with a sudden onset of flu-like symptoms. The medical staff will arrange for a nasopharyngeal swab to be performed if the subject has at least one respiratory (cough, sore throat, shortness of breath, runny nose, stuffy nose, sneezing and earache) and one systemic symptom (fever, malaise, headache and myalgia (muscle and joint pain). Swabs should be taken from the reported subjects within 3 days from the trial center being contacted or within 4 days of the onset of symptoms, whatever time is shorter.
Time Frame	For up to 4 months during the influenza season

Outcome Measure Data

Analysis Population Description

FAS: Full Analysis Set includes subjects that completed vaccination and provided samples to measure immunogenicity data prevaccination (day 0) and at least one time point post vaccination (day 42 or day 180)

Arm/Group Title	2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description:	FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Overall Number of Participants Analyzed	58	51	32	26
Measure Type: Number; Unit of Measure: percentage of subjects
positive for influenza A strains	5.2	7.8	6.3	19.2
positive for influenza B strains	0.0	2.0	3.1	3.8
positive for influenza H1 strains	0.0	0.0	0.0	0.0
positive for influenza H3 strains	5.2	7.8	6.3	19.2
positive for any influenza strain	5.2	9.8	9.4	23.1

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Differences in the infection rates against any of the strains tested between treatment group and corresponding placebo.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.662
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 2

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Differences in the infection rates against any of the strains tested between treatment group and corresponding placebo.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.168
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

12. Other Pre-specified Outcome

Title	Severity of Symptoms in RT-PCR Influenza-confirmed Subjects.
Description	Subjects recorded daily influenza symptoms from December 2016 up to March 2017. Subjects with a sudden onset of at least one respiratory and one systemic symptom were swabbed. If the results were positive for influenza then the symptoms were included in the analysis. The duration of symptoms was recorded as the number of symptomatic days during the influenza episode. Fever (≥38oC), malaise, headache, myalgia (muscle and joint pain), cough, sore throat, shortness of breath, runny nose, stuffy nose, sneezing and earache were scored on a severity scale of 0 to 3 (0: no symptom, 1: mild, 2: moderate, 3: severe). The daily severity score was the sum of the severity score for all symptoms listed above on a single day. The total score was the sum of all daily scores during the influenza episode. The peak score was the highest daily score during the influenza episode. The average score was the total score divided by the number of days the influenza episode lasted.
Time Frame	Symptoms experienced during an influenza infection episode, approximately 7-10 days.

Outcome Measure Data

Analysis Population Description

FAS: Full Analysis Set includes subjects that completed vaccination and provided samples to assess immunogenicity at pre-vaccination (day 0) and at least one post-vaccination time point (day 42 or day 180).

Arm/Group Title	2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description:	FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Overall Number of Participants Analyzed	58	51	32	26
Mean (Standard Error); Unit of Measure: units on a scale
total symptom score	113.67 (50.87)	30.80 (8.46)	57.33 (13.28)	55.17 (12.96)
total symptom peak	17.67 (3.48)	13.80 (1.16)	15.33 (1.20)	16.50 (2.73)
average severity symptom score	10.63 (1.13)	10.92 (1.61)	8.59 (1.44)	13.76 (2.12)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	[Not Specified]
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	>0.05
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]
Other Statistical Analysis		The study was not powered to detected statistical significant differences in this outcome.

Statistical Analysis 2

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	[Not Specified]
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	>0.05
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]
Other Statistical Analysis		Study not powered to detect statistically significant differences

13. Other Pre-specified Outcome

Title	Duration of Influenza Symptoms in RT-PCR Confirmed Infected Subjects.
Description	Subjects recorded daily influenza symptoms from December 2016 up to March 2017. Subjects with a sudden onset of at least one respiratory and one systemic symptom were swabbed. If the results were positive for influenza then the symptoms were included in the analysis. The duration of symptoms was recorded as the number of symptomatic days during the influenza episode. The following symptoms were recorded: fever (≥38oC), malaise, headache, myalgia (muscle and joint pain), cough, sore throat, shortness of breath, runny nose, stuffy nose, sneezing and earache.
Time Frame	During an influenza episode

Outcome Measure Data

Analysis Population Description

FAS population: subjects that completed the vaccination and had immunogenicity data at pre-vaccination and at least one time point post-vaccination.

Arm/Group Title	2x Non-adjuvanted FLU-v	1x Adjuvanted FLU-v	Non-adjuvanted Placebo	Adjuvanted Placebo
Arm/Group Description:	FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
Overall Number of Participants Analyzed	58	51	32	26
Mean (Standard Error); Unit of Measure: days
	11.67 (5.61)	3.20 (1.07)	7.33 (2.19)	4.00 (0.86)

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison in the duration of symptoms between treatment group and corresponding placebo.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.513
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 2

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the duration of symptoms between treatment group and corresponding placebo.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.578
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 3

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of total symptom score between treatment group and corresponding placebo.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.513
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 4

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the total symptom score between treatment group and corresponding placebo.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.200
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 5

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the symptom peak between treatment group and corresponding placebo.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.658
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 6

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the symptom peak between treatment group and corresponding placebo.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.640
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 7

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Non-adjuvanted Placebo
	Comments	Comparison of the average symptom score between treatment group and corresponding placebo.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.127
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

Statistical Analysis 8

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Adjuvanted Placebo
	Comments	Comparison of the average symptom score between treatment group and corresponding placebo.
	Type of Statistical Test	Other
	Comments	[Not Specified]
Statistical Test of Hypothesis	P-Value	0.201
	Comments	[Not Specified]
	Method	Wilcoxon (Mann-Whitney)
	Comments	[Not Specified]

14. Post-Hoc Outcome

Title	Effect of Influenza Vaccination in Previous 2 Years on Immunogenicity
Description	Antigen specific responders in FLU-v vaccinated arms compared to combined placebo group, grouped into those who had recieved an influenza vaccination within the previous 2 years and those who had not ever received a previous influenza vaccination. Responders were defined as those having a ≥2-fold increase in response in IFN-gamma secretion by peripheral blood mononuclear cells from day 0 to day 42 or day 180, as measured by enzyme-linked immunosorbent assay. The combined placebo group includes participants randomly assigned to adjuvanted placebo and those assigned to nonadjuvanted placebo.
Time Frame	day 0 to day 42, day 0 to day 180

Outcome Measure Data

Analysis Population Description

FAS

Arm/Group Title		2x Non-adjuvanted FLU-v, Previous Vaccination Within 2 Years	1x Adjuvanted FLU-v, Previous Vaccination Within 2 Years	Combined Placebo, Previous Vaccination Within 2 Years	2x Non-adjuvanted FLU-v, no Previous Vaccination	1x Adjuvanted FLU-v, no Previous Vaccination	Combined Placebo, no Previous Vaccination
Arm/Group Description:		participants who had received an influenza vaccination in the previous 2 years, and who received FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	participants who had received an influenza vaccination in the previous 2 years, and who received adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	participants who had received an influenza vaccination in the previous 2 years, and who received either non-adjuvanted placebo or adjuvanted placebo Non-adjuvanted Placebo: saline solution (0.5ml) on Day 0 and Day 21 Adjuvanted Placebo: Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection	Participants who had not ever previously received an influenza vaccination, and who received FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH	participants who had not ever previously received an influenza vaccination, and who received adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline	participants who had not ever previously received an influenza vaccination, and who received either non-adjuvanted placebo or adjuvanted placebo Non-adjuvanted Placebo: saline solution (0.5ml) on Day 0 and Day 21 Adjuvanted Placebo: Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21
Overall Number of Participants Analyzed		14	18	16	37	25	33
Measure Type: Count of Participants; Unit of Measure: Participants
Responders on day 42	Number Analyzed	12 participants	15 participants	13 participants	31 participants	21 participants	24 participants
		6 50.0%	13 86.7%	6 46.2%	20 64.5%	21 100.0%	10 41.7%
Responders on day 180	Number Analyzed	10 participants	15 participants	13 participants	33 participants	21 participants	25 participants
		5 50.0%	13 86.7%	8 61.5%	15 45.5%	21 100.0%	15 60.0%

Statistical Analysis 1

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Previous Vaccination Within 2 Years, Combined Placebo, Previous Vaccination Within 2 Years
	Comments	comparison between groups on day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.85
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 2

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Previous Vaccination Within 2 Years, Combined Placebo, Previous Vaccination Within 2 Years
	Comments	comparison between groups on day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.042
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 3

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, Previous Vaccination Within 2 Years, Combined Placebo, Previous Vaccination Within 2 Years
	Comments	comparison between groups on day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.69
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 4

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, Previous Vaccination Within 2 Years, Combined Placebo, Previous Vaccination Within 2 Years
	Comments	comparison between groups on day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.198
	Comments	[Not Specified]
	Method	Fisher Exact
	Comments	[Not Specified]

Statistical Analysis 5

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, no Previous Vaccination, Combined Placebo, no Previous Vaccination
	Comments	comparison between groups on day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.092
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 6

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, no Previous Vaccination, Combined Placebo, no Previous Vaccination
	Comments	comparison of groups on day 42
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 7

Statistical Analysis Overview	Comparison Group Selection	2x Non-adjuvanted FLU-v, no Previous Vaccination, Combined Placebo, no Previous Vaccination
	Comments	comparison of groups on day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	0.27
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Statistical Analysis 8

Statistical Analysis Overview	Comparison Group Selection	1x Adjuvanted FLU-v, no Previous Vaccination, Combined Placebo, no Previous Vaccination
	Comments	comparisons between groups on day 180
	Type of Statistical Test	Other
	Comments	inequality test
Statistical Test of Hypothesis	P-Value	<0.001
	Comments	[Not Specified]
	Method	Chi-squared
	Comments	[Not Specified]

Adverse Events

Time Frame	From vaccination to study completion (approx. 7 months)
Adverse Event Reporting Description	Solicited Adverse Events were collected for 21 days after each vaccination. Subjects had to fill in the AEs diary card daily and return to the clinic on the next scheduled visit. Unsolicited Adverse Events and Severe Adverse Events were collected at any time during the study directly to the PI or study doctor.
Arm/Group Title	2x Non-adjuvanted FLU-v		1x Adjuvanted FLU-v		Non-adjuvanted Placebo		Adjuvanted Placebo
Arm/Group Description	FLU-v on Day 0 and Day 21 FLU-v: Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH		adjuvanted FLU-v on Day 0, saline (0.5mL) on Day 21 adjuvanted FLU-v: Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection Saline: Subcutaneous injection in the upper arm with 0.5ml of saline		saline solution (0.5ml) on Day 0 and Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline		Adjuvanted placebo on Day 0, saline (0.5mL) on Day 21 Saline: Subcutaneous injection in the upper arm with 0.5ml of saline Adjuvanted placebo: Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection
All-Cause Mortality
	2x Non-adjuvanted FLU-v		1x Adjuvanted FLU-v		Non-adjuvanted Placebo		Adjuvanted Placebo
	Affected / at Risk (%)		Affected / at Risk (%)		Affected / at Risk (%)		Affected / at Risk (%)
Total	0/58 (0.00%)		0/57 (0.00%)		0/32 (0.00%)		0/27 (0.00%)
Serious Adverse Events
	2x Non-adjuvanted FLU-v		1x Adjuvanted FLU-v		Non-adjuvanted Placebo		Adjuvanted Placebo
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	2/58 (3.45%)		2/57 (3.51%)		0/32 (0.00%)		0/27 (0.00%)
Cardiac disorders
Myocardial Infarction * 1	1/58 (1.72%)	1	0/57 (0.00%)	0	0/32 (0.00%)	0	0/27 (0.00%)	0
Injury, poisoning and procedural complications
upper limb fracture * 1	0/58 (0.00%)	0	1/57 (1.75%)	1	0/32 (0.00%)	0	0/27 (0.00%)	0
Psychiatric disorders
Depression * 1	0/58 (0.00%)	0	1/57 (1.75%)	1	0/32 (0.00%)	0	0/27 (0.00%)	0
Alcohol problem * 1	0/58 (0.00%)	0	1/57 (1.75%)	1	0/32 (0.00%)	0	0/27 (0.00%)	0
Surgical and medical procedures
Abdominal hernia repair * 1	1/58 (1.72%)	1	0/57 (0.00%)	0	0/32 (0.00%)	0	0/27 (0.00%)	0
1 Term from vocabulary, MedDRA (Unspecified); * Indicates events were collected by non-systematic assessment
Other (Not Including Serious) Adverse Events
Frequency Threshold for Reporting Other Adverse Events	5%
	2x Non-adjuvanted FLU-v		1x Adjuvanted FLU-v		Non-adjuvanted Placebo		Adjuvanted Placebo
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	41/58 (70.69%)		50/57 (87.72%)		27/32 (84.38%)		24/27 (88.89%)
Blood and lymphatic system disorders
Lymphadenopathy † 1	7/58 (12.07%)	8	4/57 (7.02%)	4	3/32 (9.38%)	4	4/27 (14.81%)	4
Gastrointestinal disorders
diarrhoea † 1	6/58 (10.34%)	8	8/57 (14.04%)	9	4/32 (12.50%)	4	2/27 (7.41%)	2
Vomiting † 1	3/58 (5.17%)	3	5/57 (8.77%)	6	2/32 (6.25%)	2	0/27 (0.00%)	0
Abdominal pain † 1	7/58 (12.07%)	7	7/57 (12.28%)	8	1/32 (3.13%)	2	2/27 (7.41%)	2
General disorders
Fatigue † 1	14/58 (24.14%)	16	17/57 (29.82%)	25	11/32 (34.38%)	12	6/27 (22.22%)	6
Influenza like illness * 1	11/58 (18.97%)	12	18/57 (31.58%)	19	10/32 (31.25%)	11	9/27 (33.33%)	12
Injection site erythema † 1	13/58 (22.41%)	15	38/57 (66.67%)	40	0/32 (0.00%)	0	6/27 (22.22%)	6
Injection site pain † 1	4/58 (6.90%)	6	39/57 (68.42%)	46	4/32 (12.50%)	4	11/27 (40.74%)	12
Injection site swelling † 1	6/58 (10.34%)	7	38/57 (66.67%)	39	0/32 (0.00%)	0	7/27 (25.93%)	7
Injection site warmth † 1	7/58 (12.07%)	9	29/57 (50.88%)	30	1/32 (3.13%)	1	6/27 (22.22%)	7
Injection site pruritus † 1	9/58 (15.52%)	10	22/57 (38.60%)	23	0/32 (0.00%)	0	10/27 (37.04%)	14
Injection site induration † 1	16/58 (27.59%)	18	49/57 (85.96%)	49	0/32 (0.00%)	0	10/27 (37.04%)	11
Haematoma at Injection Site † 1	5/58 (8.62%)	6	19/57 (33.33%)	19	0/32 (0.00%)	0	5/27 (18.52%)	5
Infections and infestations
Upper respiratory tract infection * 1	14/58 (24.14%)	15	14/57 (24.56%)	15	13/32 (40.63%)	14	6/27 (22.22%)	6
Herpes simplex † 1	2/58 (3.45%)	2	2/57 (3.51%)	2	3/32 (9.38%)	3	1/27 (3.70%)	1
Metabolism and nutrition disorders
Decreased apetite † 1	1/58 (1.72%)	1	11/57 (19.30%)	12	0/32 (0.00%)	0	1/27 (3.70%)	1
Musculoskeletal and connective tissue disorders
arthralgia † 1	4/58 (6.90%)	5	5/57 (8.77%)	6	1/32 (3.13%)	1	0/27 (0.00%)	0
Back pain † 1	5/58 (8.62%)	5	5/57 (8.77%)	7	6/32 (18.75%)	7	2/27 (7.41%)	2