Downloaded from 8 9 10 · 93 the WHO manual of Animal Influenza Diagnosis a nd Surveillance 2002 (11). Briefly, serial ten-94 fold dilutions of virus-containing daily post-cha llenge

Variations in disease severity outcomes for Influenza live viral challenges in man: Meta-1

analysis and potential role of pre-existing heterosubtypic cellular immunity 2

Running Title: Cellular immunity and outcomes of live influenza challenges 3

Olga PLEGUEZUELOS, Stuart ROBINSON, Ana FERNANDEZ, Gregory A. 4

STOLOFF, and Wilson CAPARRÓS-WANDERLEY*. 5

Affiliations: 6

SEEK, Central Point, 45 Beech Street, London, EC2Y 8AD, United Kingdom 7

(*) Corresponding author: 8

Dr Wilson Caparrós-Wanderley 9

SEEK, Central Point, 45 Beech Street, London EC2Y 8AD 10

Email: [email protected] 11

Tel: +44 (0)20-71536570 12

13

CVI Accepted Manuscript Posted Online 17 June 2015Clin. Vaccine Immunol. doi:10.1128/CVI.00101-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 14

Objectives: Live Influenza challenges in man are valuable models for testing the efficacy of 15

vaccines and antiviral agents. Volunteers are treated with an investigational agent and their 16

clinical outcomes post-challenge are compared to those of Placebo treated volunteers. Despite 17

using a common protocol, recruitment criteria and a similar dose of the same challenge strain, 18

we noticed differences in disease severity outcomes between Placebo groups from different 19

studies. We investigated whether these differences were significant and, if so, whether any 20

pattern was identifiable and its possible causes. 21

Methods: We compared the clinical outcomes post-challenge in Placebo groups from five 22

clinical studies carried out between 2008 and 2013. Correlations between pre-challenge 23

heterosubtypic cellular response (IFN−γ) and post-challenge clinical outcomes were also 24

investigated in one study. 25

Results: Placebo groups from studies carried out between 2009 and 2010 attained significantly 26

reduced (p<0.05) symptom scores post-challenge than Placebo groups from studies carried out 27

in either 2008 or 2013. Also, in a 2010 study, the frequency of high influenza heterosubtypic 28

cellular responders pre-vaccination was significantly lower in the test group (FLU-v) than in the 29

Placebo group (p=0.04). Moreover, the Placebo group’s increased pre-existing heterosubtypic 30

cellular response correlated with reductions in symptom score and viral shedding post-31

challenge (p≤0.023). Only post-vaccination did the Test group display an equivalent correlation. 32

Conclusions: The last influenza pandemic coincides with a significant reduction in disease 33

severity outcomes. This reduction also appears to correlate with increased pre-existing 34

influenza heterosubtypic cellular responses. 35


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INTRODUCTION 36

Live influenza challenges in man are valuable models for testing the efficacy of vaccines and 37

antiviral agents. Their basis is simple: a group of volunteers is treated with an investigational 38

agent and their clinical outcomes post-challenge are compared to those of a group of Placebo 39

treated volunteers. Their logistics, in contrast, are complex. 40

Influenza infection elicits a range of immune responses. One such response is the production of 41

strain-specific neutralising antibodies that confer immunity against infection by the same strain 42

(1). As a result, a key volunteer exclusion criterion in challenge studies is the detection of pre-43

existing neutralising antibodies (HAI>10) to the challenge strain. Another such response is the 44

generation of antiviral cellular immune responses. Despite existing evidence as to their 45

protective role during infection (2-4), pre-existing cellular immune responses to the challenge 46

strain are not normally assessed during volunteer recruitment. 47

We have developed a novel vaccine (FLU-v) that elicits broad influenza heterosubtypic cellular 48

responses without inducing any significant antibody response (5,6, 7). In humans, FLU-v was 49

found to be safe, well tolerated and, in a live challenge study, to induce a vaccine specific 50

cellular response whose magnitude correlated with reductions in symptom score and viral 51

shedding (7). No such correlations were seen in the Placebo group, but we did notice that both 52

viral shedding and symptom score post-challenge were much lower (50%) in our Placebo group 53

than in the Placebo group from a previous study. To establish the significance of these 54

differences, we compared the Placebo group outcomes of several other live influenza challenge 55

studies. All these studies, although involving different placebo agents, were carried out by the 56

same clinical group by the same clinical group (Retroscreen Ltd), using the same recruitment 57


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criteria, the same viral strain and dose and the same method for determining post-challenge 58

clinical and virological outcomes. This meta-analysis revealed an experiment of nature that we 59

believe provides interesting insights in the potential of the cellular immune system in 60

controlling Influenza infection. 61

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MATERIALS AND METHODS 63

Clinical trial data used for meta-analysis 64

The reported clinical outcomes post-challenge for the Placebo group of four reported 65

independent clinical trials (3, 7, 8, 9) and one previously unreported study (Retroscreen Ltd, 66

personal communication) were used for the meta-analysis. The Placebo agent used in each 67

study was different, but all the studies were carried out by the same clinical group (Retroscreen 68

Ltd) and followed a common challenge protocol (Fig. 1) that used the same well defined 69

recruitment criteria, viral challenge strain (A/Wisconsin/67/2005, H3N2) and procedures for the 70

assessment of disease severity and viral shedding. Exact details for each study are provided in 71

the referenced manuscripts, but they are also briefly summarised below. 72

Recruitment criteria and study procedures: Healthy male subjects aged 18 to ~45 years with no 73

clinically significant abnormal findings (i.e. physical examination, medical history or laboratory 74

results) and no medical history of Influenza-like illness in the prior 12 months were assessed for 75

enrolment. Only those with HAI ≤10 for the Influenza challenge strain were enrolled. 76

Following recruitment and treatment (Placebo or test agent), volunteers were challenged on 77

Day 0 by nasal instillation with 1 ml of solution containing approximately 105.25 50% tissue 78

infective dose per ml of live A/Wisconsin/67/2005 (H3N2, tissue culture grown). From days 5 to 79

7 volunteers received antiviral treatment (e.g. oseltamivir) before being released from 80

quarantine on Day 7. 81

Physical examinations and clinical laboratory tests were performed at screening, pre- and post-82

treatment (both pre-challenge), and daily from Day -2 pre-challenge to Day 7 post-challenge. A 83

final assessment was carried out around Day 28 post-challenge. Volunteer self-recorded 84


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observations pre- and post-challenge, as well as the scripted symptom questionnaires, were 85

assessed by clinical staff. 86

Symptom scoring and virology and HAI tests: Symptom score was determined using a 87

standardised scoring system (3,10) based on subject self-assessment and clinician’s 88

examination. A range of parameters (e.g. Runny Nose, Stuffy Nose, Sneezing, Sore Throat, 89

Earache, Malaise, Cough, Shortness of Breath, Headache and Muscle/Joint ache) were assessed 90

and scored from 0 to 3, corresponding to from absent to severe. 91

Viral shedding in the nasopharyngeal samples was determined by TCID50 assay as described in 92

the WHO manual of Animal Influenza Diagnosis and Surveillance 2002 (11). Briefly, serial ten-93

fold dilutions of virus-containing daily post-challenge nasal lavage samples were inoculated into 94

96-well microtitre plates seeded with Madin-Darby canine kidney (MDCK) cells. Cytopathic 95

effects in individual wells were determined after 5–6 days incubation at 37°C. Viral shedding 96

was defined as a viral culture titre greater than 1.5 log10 TCID50/ml. 97

Haemagglutinin-specific antibody titres against the challenge virus in volunteer’s sera samples 98

were determined by HAI assay using chicken erythrocytes as described in the WHO manual of 99

Animal Influenza Diagnosis and Surveillance 2002 (11). 100

Regulatory Approval and Ethical Considerations: 101

All studies included in the meta-analysis were reported as conducted in accordance with Good 102

Clinical Practice, the Declaration of Helsinki (1964 and 2008), and all regulatory requirements. 103

As we are also reporting previously undisclosed experimental data, we confirm that our FLU-v 104

study (7) was approved by the Plymouth Independent Ethics Committee under REC reference 105


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number 10/IEC04/1. The trial was registered under EUDRA Identifier 2009-014716-35 and 106

NCT01226758. Written informed consent was obtained from all participants. 107

Vaccine description 108

FLU-v is a sterile equimolar mixture of four polypeptides encoding immunoreactive conserved 109

regions within Influenza (5,6, 7). These sequences, which are shown below, were synthetically 110

manufactured (Bachem AG, Bübendorf, Switzerland) in accordance with current Good 111

Manufacturing Practice. 112

M1 - DLEALMEWLKTRPILSPLTKGILGFVFTLTVP (32 aa) 113

NPA - DLIFLARSALILRGSVAHKSC (21 aa) 114

NPB - PGIADIEDLTLLARSMVVVR (20 aa) 115

M2 - IIGILHLILWILDRLFFKCIYRLF (24 aa) 116

FLU-v was administered subcutaneously in 1.0 ml volume as a single 500 μg dose in saline 117

emulsified (1:1) with adjuvant ISA-51 (Seppic, France). The Placebo was saline emulsified with 118

ISA-51. The adjuvant is composed of a light mineral oil and a surfactant system designed to 119

make a water-in-oil emulsion. Functionally, ISA-51 is not known to preferentially favour the 120

induction of Th1-like responses (12). 121

Heterosubtypic Cellular immunity – Cytokine ELISA 122

Blood was harvested pre-challenge on days -21 (i.e. pre-vaccination) and -2 (i.e. 19 days post-123

vaccination) and PBMCs isolated and frozen. Thawed PBMCs were seeded at 2x105 cells/well 124

(96 well plate) in RPMI-1640 (Sigma, UK), supplemented with 25 mM HEPES, Penicillin (100 125

units/ml), Streptomycin (100 μg/ml) and 10% FCS, and containing one of the following test 126

antigens: 1 μg/ml Con A (Sigma, UK), 1 μg/ml Bovine Serum Albumin (BSA; Sigma, UK) or live 127


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Influenza A/Swine/Iowa/15/30 (H1N1, Multiplicity of Infection 10). Virus (egg grown) was 128

obtained from NIBSC as low-endotoxin preparations suitable for in vitro cellular analysis. Each 129

antigen was tested in triplicate. After 24 h incubation at 37oC and 5% CO2, IFN−γ production in 130

the cell supernatant for each of the test antigens was determined using a validated ELISA assay 131

(BD, UK; Human IFN−γ kit 555142). Response levels were calculated as pg/ml of IFN−γ produced 132

against a standard provided with the assay kit. Minimum level of detection for the assay is 9 133

pg/ml IFN−γ. 134

Strong heterosubtypic cellular responses were defined as those in which an individual’s IFN−γ 135

response to the Influenza A/Swine/Iowa/15/30 (H1N1) virus were ≥4-fold higher than the 136

individual’s IFN−γ response to the negative control (i.e. BSA + medium). 137

Statistical analysis 138

Inter-group differences in total mean symptom score and viral shedding were determined by 139

single-factor ANOVA analysis. Pairwise differences between the studies were determined by T-140

test (2-way), with adjustment of significance for multiple pairwise comparisons by the Tukey-141

Kramer HSD (Honest Significant Difference) method. Heterosubtypic responder frequencies 142

were analysed by the Friedman Exact test, whilst correlations between clinical outcomes and 143

heterosubtypic cellular response levels were determined using the Spearman Rank correlation 144

test. 145

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RESULTS 147

Mean total symptom score post-challenge: inter-study variability 148

We have previously reported (7) how in an Influenza live challenge study carried out in 2010, 149

vaccination with FLU-v induced an IFN−γ response to the vaccine whose magnitude correlated 150

with reductions in both viral titre (p=0.01) and total symptom score (p=0.02). No such 151

correlation was seen in the Placebo group. Although we saw no significant differences in mean 152

total symptom score post-challenge between the FLU-v and Placebo group, we did noticed a 153

significant reduction in mean total symptom score post-challenge in our 2010 Placebo group 154

compared to the Placebo group of a previous unreported study carried out by Retroscreen in 155

2008 (mean total symptom score: 11.4±13.0 vs 37.1±27.5, AVR±SD, our Placebo vs 2008 156

Placebo, p=0.006). 157

This significant difference in outcomes was surprising to us because, although the nature of the 158

Placebo agent was different in the two trials, the historical 2008 Placebo dataset (n=12) had 159

been obtained by the same clinical group (Retroscreen Ltd), using the same recruitment 160

criteria, the same viral strain and dose and the same method for determining symptom score. 161

More importantly, the outcomes of this 2008 Placebo constituted the baseline data used to 162

calculate the sample size required to meet the endpoints of our trial. 163

This difference in outcomes also raised the question of whether our observation was unique, or 164

significant differences in outcome were a common observation in live challenge studies. To 165

address this question we compared the outcome of both our 2010 Placebo and the Retroscreen 166

2008 Placebo against those reported for Placebo groups in other published studies carried out 167


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in 2008 (3), 2009 (9) and 2013 (8). These studies followed the same common standard protocol 168

(Fig. 1), recruitment criteria and procedures used in the 2010 and 2008 Placebos. 169

Statistical analysis (ANOVA) of these five studies (Table 1) revealed a significant difference 170

(p=0.004) in the mean total symptom score of the Placebo groups. Subsequent pairwise 171

comparisons (T-test with Tukey-Kramer’s HSD adjustment for significance) revealed that the 172

mean total symptom score for the Placebo Group in the 2008 Wilkinson study (3) was 173

significantly higher than that seen in our 2010 Placebo (60.8±10.7 vs 11.4±13.0, p=0.000), but 174

not different to that in the Retroscreen’s 2008 Placebo (60.8±10.7 vs 37.1±27.5, p>0.050). In 175

contrast, the mean total symptom score for the Placebo Group in the 2009 Lillie study (9) was 176

significantly lower than that seen in both the Retroscreen’s 2008 Placebo (15.3±15.1 vs 177

37.1±27.5, p=0.030) and the 2008 Wilkinson Placebo (15.3±15.1 vs 60.8±10.7, p=0.000), but not 178

different to that in our 2010 Placebo (15.3±15.1 vs 11.4±13.0, p>0.050). A final comparison of 179

these four different Placebo groups to that of the Placebo group in the 2013 Ramos’ study (8) 180

(55.5±54.8) reveals that mean total symptom score in this study is not different to that seen in 181

either the Wilkinson’s 2008 Placebo or the Retroscreen’s 2008 Placebo (p>0.050 for both), but 182

it is significantly higher than that seen in both Lillie’s 2009 Placebo (p=0.022) and our 2010 183

Placebo (p=0.007). These results indicate that, following influenza live challenge, mean total 184

symptom scores in Placebo group volunteers were significantly lower in 2009-2010 than they 185

were in either 2008 or 2013. 186

Mean total viral shedding post-challenge: inter-study variability 187

We then proceeded to test whether the observed differences in mean total symptom score 188

across the studies were also reflected in the mean total viral shedding measurements. Total 189


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viral shedding data was not reported in the Wilkinson study (3) and hence we could not include 190

this study in the analysis. Nonetheless, a comparison of the remaining four studies revealed a 191

significant difference (p=0.040) in mean total viral shedding. 192

Subsequent pairwise analysis revealed that, as shown in Table 1, mean total viral shedding in 193

the Retroscreen’s 2008 Placebo (10.1±2.9, AVR±SD) was significantly higher than in Lillie’s 2009 194

Placebo (3.3±4.3, p=0.012), our 2010 Placebo (4.0±4.4, p=0.022) and Ramos’ 2013 Placebo 195

(3.2±4.5, p=0.006). 196

Heterosubtypic immunity 197

In an attempt to determine the possible reasons for the differences amongst the groups, we 198

first analysed the infection rate for each of the studies. Infection rate was defined as the 199

percentage of volunteers with at least one positive result by TCID50 between days 1 and 5 after 200

live influenza challenge. As shown in Table 1, and despite the wide range of values, no statistical 201

differences (p>0.05) were found in the infection rates across the different studies: Wilkinson’s 202

2008 Placebo (100%), Retroscreen’s 2008 Placebo (66.6%), Lillie’s 2009 Placebo (45.5%), our 203

2010 Placebo (61.5%) and Ramos’ 2013 Placebo (48.4%). 204

The similarity in infection rate across the studies suggests that the mechanism responsible for 205

the differences in outcomes is most likely a post-infection mechanism. If correct, this would 206

exclude neutralising antibody responses, but not cellular immune responses. Unfortunately, 207

cellular responses to the challenge virus were not assessed in any of these studies, either pre- 208

or post-challenge. Moreover, if any cellular responses were measured, the antigen (e.g. virus or 209

vaccine) and the method of analysis (e.g. ELISA or ELISPOT) used were all different, thus 210

rendering any direct comparison impossible. 211


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As stated earlier, we have previously established (7) that cellular responses to a vaccine 212

correlated with reductions in both viral load and symptom score. Since the period of reduced 213

total mean symptom scores and viral shedding (2009-2010) identified from our earlier meta-214

analysis coincided with the dates of the last influenza pandemic, we decided to test if in our 215

study strong pre-existing heterosubtypic cellular responses to a H1N1 swine influenza strain 216

were common and, if so, whether their intensity negatively correlated with symptom score and 217

viral shedding. Ideally, we would have prefered to use the pandemic Influenza 218

A/California/7/2009 (H1N1) strain, but WHO recommends the use of biosafety level 2 plus 219

[BSL-2 plus] facilities with biosafety level 3 [BSL-3] practices with this strain (13). As these 220

facilities are not available to us, we settled for a BSL-2 swine strain: A/Swine/Iowa/15/30 221

(H1N1). 222

In our 2010 study, we had dosed and challenged a total of twenty-eight volunteers. However, 223

for this post-hoc analysis, frozen PMBC samples were available from only fifteen volunteers 224

(seven from the Placebo group and eight from the FLU-v group). We found (Table 2) strong pre-225

vaccination IFN−γ responses to the recall A/Swine/Iowa/15/30 (H1N1) virus (i.e. ≥4-fold IFN−γ 226

response to negative control) in all but one of the Placebo volunteers (median 7.0 fold-227

increase). In contrast, in the vaccinated (FLU-v) group, strong pre-vaccination IFN−γ response to 228

the recall A/Swine/Iowa/15/30 (H1N1) virus (median 3.4 fold-increase) were found in only one 229

volunteer. Post-vaccination, the frequency of strong IFN−γ responders became similar in both 230

groups (5 vs 4, Placebo vs FLU-v), but the overall level of IFN−γ response to the recall swine 231

virus still remained higher in the Placebo group (median 10.3 vs 5.2, Placebo vs FLU-v). 232


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Correlation analysis revealed a significant negative correlation in the Placebo group between 233

the intensity of the heterosubtypic IFN−γ response to the A/Swine/Iowa/15/30 (H1N1) virus 234

and both the mean total symptom score (r=-0.771, p=0.036; Fig 2a) and the mean total viral 235

shedding (r=-0.768, p=0.022; Fig 2b). In the FLU-v group, no significant correlations were seen 236

pre-vaccination (p>0.05), but a significant negative correlation was established post-vaccination 237

between the intensity of the heterosubtypic IFN−γ response to the A/Swine/Iowa/15/30 (H1N1) 238

virus and the mean total symptom score (r=-0.667, p=0.035; Fig 2c) 239

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DISCUSSION 242

Influenza infection elicits a range of natural antibody and cellular immune responses to the 243

virus. Some of these responses are specific to the infecting viral strain (homosubtypic 244

responses), whilst others are cross-reactive to other viral strains (heterosubtypic responses). 245

Amongst the homosubtypic responses, neutralising antibodies directed to the Haemagglutinin 246

(HA) and Neuramidase (NA) antigens are of particularly interest. Infection by one influenza 247

strain elicits neutralising HA/NA antibody responses that confer lifelong immunity against 248

infection by the same strain (1). For over 50 years influenza public health programs worldwide 249

have built upon this observation by using vaccines that induce homosubtypic neutralising 250

HA/NA antibody responses. Heterosubtypic responses, despite increasing evidence of their 251

potential protective role during infection at both the antibody (14-16) and cellular level (2-4), 252

have not yet been successfully exploited in the clinic. 253

Notwithstanding their universal use, HA/NA based vaccines suffer from major shortcomings. As 254

new variants of the virus emerge every year, the new circulating viral strains must be first 255

identified before new formulations of the vaccine are prepared every year, which in turn means 256

that every year the population must be re-vaccinated (17). A clear need remains for a vaccine 257

that can address these shortcomings. 258

Live challenge studies in man are valuable models for the development of effective therapies 259

(e.g. vaccines and antivirals) against Influenza. They allow the efficacy of a candidate treatment 260

to be assessed by comparing disease severity outcomes post-challenge between volunteer 261

groups treated with either the candidate therapy or a Placebo. 262


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Recognising the importance of neutralising antibody responses in influenza protection, the 263

identification of pre-existing neutralising antibody titres (i.e. HAI>10) to the challenge strain is a 264

key universal exclusion criterion during volunteer recruitment in live challenge studies (3, 7, 8, 265

9). In contrast, neither pre-existing heterosubtypic immune responses (antibody or cellular) nor 266

pre-existing homosubtypic cellular responses to the challenge strain are regularly assessed 267

during volunteer recruitment. 268

In 2010 we carried out a live challenge study in humans using a novel vaccine (FLU-v) designed 269

to elicit cellular immune responses against influenza. Despite induction of a FLU-v specific IFN−γ 270

response (6, 7) that correlated with reductions in viral shedding and symptom score (7), no 271

significant differences in clinical outcome was seen between the Placebo and FLU-v groups. 272

However, we did identify a clear and significant reduction in both viral shedding and symptom 273

score in our 2010 Placebo group compared to a Placebo group from a study carried out by 274

Retroscreen in 2008. This 2008 Placebo group is significant because its outcomes constitute the 275

baseline data used to calculate the sample size needed to meet the endpoints of our 2010 trial. 276

As a certain degree of variability is expected in all biological systems, we decided to investigate 277

how consistent viral shedding and symptom score outcomes were across five live challenge 278

studies carried out between 2008 and 2013. Meta-analysis of historical data is extensively used 279

in clinical research (18-23) and, under certain stringent rules, is even allowed by both FDA and 280

EMEA to assess the efficacy of a treatment (24,25). These rules state that all the data analysed 281

must come from clinical trials that used the same eligibility criteria, measured comparable 282

variables and were carried out by the same investigators. Since all the five clinical studies 283

considered in our meta-analysis were carried out by the same clinical group (Retroscreen Ltd), 284


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using the same well defined recruitment criteria, following a common challenge protocol that 285

used a similar dose of the same viral strain (A/Wisconsin/67/2005, H3N2) and assessed the 286

same parameters (i.e. symptom score and viral shedding), we were confident of the validity of 287

our approach. Of course, the nature of the Placebo in these five studies was different, but since 288

we and Retroscreen Ltd agreed to use historical data from the 2008 Placebo group to 289

determine the required sample size of our 2010 study, we believe this decision was consistent 290

with and supports our multi-study comparative approach. 291

The meta-analysis revealed that of the five studies analysed, the two carried out between 2009 292

and 2010 (7, 9) achieved total mean symptom scores post-challenge that were significantly 293

lower (~50%) than those seen in studies carried out in either 2008 (3) or 2013 (8). Viral 294

shedding was also significantly higher in the 2008 studies than in the 2009 and 2010 studies, 295

and, in contrast to symptom score, it was also higher than in the 2013 study. 296

An accurate determination of the mechanism(s) responsible for these differences was not 297

possible as the immune/pharmacological effector mechanisms assessed were different for each 298

study. However, because (a) infection rates (determined as the percentage of challenged 299

volunteers that develop a positive TCID50 between days 1 and 5 post-challenge) across all five 300

studies were not statistically different, (b) neutralising antibodies act primarily at the pre-301

infection stage, and (c) all volunteers had HAI titres to the challenge strain ≤10, we do not 302

believe that the observed inter-study differences were caused by differences in the volunteers’ 303

HAI titres. 304

Assessment of cellular responses amongst the studies was not possible. Pre-existing cellular 305

responses to the challenge virus are not regularly assessed in these studies and, when cellular 306


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responses are measured, the antigen (e.g. virus or vaccine) and the method of analysis (e.g. 307

ELISA or ELISPOT) used are all different, thus rendering any direct comparison impossible. 308

Nonetheless, three observations lead us to consider the possibility that differences in the pre-309

existing influenza heterosubtypic cellular responses may be, at least partially, responsible for 310

the observed inter-study differences in Placebo group outcomes post-challenge. Firstly, the two 311

studies showing significant reductions in mean total symptom scores were those carried in 312

2009-2010. These dates coincide with the last influenza pandemic. Secondly, we (7) and others 313

(3, 4) have shown significant negative correlations between the intensity of the cellular 314

response and measurements of influenza disease severity. Thirdly, the reduction in viral 315

shedding, but not in either symptom score or rate of infection, in the 2013 study compared to 316

the 2008 Placebo suggests that a post-infection mechanism is controlling viral replication. 317

Although lack of data prevented us from comparing the role of heterosubtypic cellular 318

responses across the five studies considered, we believed that some relevant evidence could 319

still be obtained through additional testing of PBMC samples from our 2010 study. 320

Unfortunately, we did not have a complete sample set for this post-hoc analysis and hence we 321

accept that the small size of the sample (15 individuals) further limits the power of this analysis. 322

Nonetheless, we found significant correlations between the pre-existing IFN−γ responses to 323

influenza A/Swine/Iowa/15/30 (H1N1) and reductions in both total mean symptom score and 324

total mean viral shedding in the Placebo group. 325

An additional and surprising finding of our analysis was that the frequency of pre-existing high 326

IFN−γ responders to A/Swine/Iowa/15/30 (H1N1) was much higher in the Placebo group than in 327

the vaccine (FLU-v) group. Moreover, although no significant correlation between the IFN−γ 328


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response to the influenza A/Swine/Iowa/15/30 (H1N1) strain and reductions in viral shedding 329

was seen pre-vaccination in the FLU-v group, this correlation became evident post-vaccination. 330

Of course, we have no evidence that the pattern of heterosubtypic cellular responses (i.e. to 331

A/Swine/Iowa/15/30, H1N1) is the same as that of the homosubtypic cellular responses (i.e. to 332

the challenge strain A/Wisconsin/67/2005, H3N2). However, we would maintain that it is not 333

unreasonable to expect it to be so. 334

We have no explanation as to how, despite the randomisation and double-blind nature of the 335

study, our Placebo group ended up with a higher number of volunteers with strong 336

heterosubtypic cellular responses than our FLU-v group. Recruitment criteria and 337

randomisation in our study was not different to the other studies included in our meta-analysis. 338

A post-hoc analysis of pre-existing HAI responses in our volunteers to the actual 2009-2010 339

pandemic strain (A/California/7/2009, H1N1) did not reveal any positive individual in either the 340

Placebo or the FLU-v group (data not shown). Nonetheless, we cannot completely rule out a 341

difference in the exposure rate to the virus between the two groups. A report by Presanis et al 342

(26) suggests that the rate of asymptomatic infection in England during the pandemic [June 343

2009 to February 2010] was as high as 65%. With the benefit of hindsight, and since our study 344

took place shortly after the end of the pandemic, it is our opinion that the list of exclusion 345

criteria used (i.e. history of Influenza-like illness over the previous 12 months and HAI > 10) was 346

ill suited to prevent the recruitment of asymptomatically infected individuals. 347

Increased levels of influenza heterosubtypic cellular responses in the population after the 348

pandemic, could also help to explain the particular results of the Placebo group in the Ramos’ 349

2013 study. McMichael et al (27) showed that T cell responses to influenza are detectable years 350


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after initial natural exposure, although their number declines rapidly with time. As T cell 351

responses are widely acknowledged to play a key anti-viral role, it is possible that exposure to 352

the challenge virus may have caused the expansion of a small pool of memory influenza 353

heretosubtypic T cell clones. The expansion of this small population may not have been 354

sufficient to significantly reduce symptom severity (total symptom score), but it may have been 355

able to have a negative effect on the rate of viral proliferation (total viral shedding). 356

In summary, we believe our results provide evidence of an unplanned “experiment of nature” 357

that adds to the existing body of evidence on the ability of heterosubtypic cellular immunity to 358

reduce influenza disease severity in humans (2-4). As such, it supports our efforts, and those of 359

other groups, in developing vaccines that elicit heterosubtypic cellular immune responses 360

against influenza. Whether it constitutes sufficient evidence to justify the consistent screening 361

of volunteers for pre-existing cellular immunity to the challenge strain during recruitment, we 362

leave that decision to any researcher planning to use live influenza challenge models in man in 363

the future. 364

365


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Table 1 455

Summary of descriptive statistics for the post-challenge outcome of all analysed studies. Total 456

symptom score is the sum of all measured symptoms scores for an individual from day 1 to day 457

7 following challenge with influenza A/Wisconsin/67/2005 (H3N2). Infection rates are the 458

percentage of challenged volunteers with at least one daily nasal sample positive for influenza 459

A/Wisconsin/67/2005 (H3N2) post-challenge. Total viral shedding represents the sum of all 460

measured viral shedding for an individual from day 1 to day 5 post-challenge with influenza 461

A/Wisconsin/67/2005 (H3N2). Viral shedding on days 6 and 7 post-challenge is not considered 462

as under the clinical protocol used all individuals receive anti-viral treatment (e.g. oseltamivir) 463

on those days. Abbreviations: Average (AVR), Standard Deviation (STDEV), Lowest value in 464

range (min), Highest value in range (max). N/A indicates that no data is available. 465

466

Table 2 467

Heterosubtypic cellular immune responses pre- and post-vaccination in 2010 FLU-v study. 468

Values are represented as the fold-increase in IFN−γ response to A/Swine/Iowa/15/30 (H1N1) 469

compared to the negative control. IFN−γ (pg/ml) responses to the negative control pre- and 470

post-vaccination for both groups are 99.2±25.7 vs 80.6±12.9 pg/ml. IFN−γ (pg/ml) response to 471

the positive control (ConA) pre- and post-vaccination for both groups are 311±85 vs 378±35 472

pg/ml. 473

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Figure 1 475

Consort profile. Trial profile and baseline demographic data for enrolled volunteers in all five 476

studies analysed. The reported median age of the volunteers in the studies ranged from 24 to 477

30 years. Where this information is provided, studies are reported to have been carried out 478

between August and November. The section in grey refers to data not incorporated in the 479

meta-analysis of Placebo groups, but that was used for the comparison of cellular immunity 480

described later on in the manuscript. 481

482

Figure 2 483

Correlation analysis between heterosubtypic cellular responses and measurements of disease 484

severity post-challenge. Values are represented as the fold-increase in IFN−γ response to 485

A/Swine/Iowa/15/30 (H1N1) compared to the negative control. Figures 2A and 2B represent 486

the correlations between the Placebo group’s pre-existing heterosubtypic cellular response 487

and, respectively, its mean total symptom score and mean total viral shedding post-challenge. 488

Figure 2C represents the correlation between the FLU-v group’s post-vaccination 489

heterosubtypic cellular response and its mean total symptom score post-challenge. All analyses 490

were carried out using the Spearman rank correlation test. 491


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A B C D E

Retroscreen Wilkinson et al Lillie et al FLU-v study Ramos et al

2008 Placebo 2008 Placebo 2009 Placebo 2010 Placebo 2013 Placebo

n 12 4 11 13 31

AVR 37.1 60.8 15.3 11.4 55.5

STDEV 27.5 10.7 15.1 13.0 54.8

MEDIAN 50.0 57.5 8.0 8.0 39.0

min 0.0 52.0 0.0 0.0 0.0

max 74.0 76.0 38.0 44.0 190.0

A

B p > 0.050

C p = 0.030 p = 0.000

D p = 0.006 p = 0.000 p > 0.050

E p > 0.050 p > 0.050 p = 0.022 p = 0.007

TOTAL

SYMPTOM

SCORE

ANOVA

PAIRWISE

COMPARISON

T-TEST

p = 0.004

E p > 0.050 p > 0.050 p = 0.022 p = 0.007

66.7% 100.0% 45.5% 61.5% 48.4%

AVR 10.1 n/a 3.3 4.0 3.2

STDEV 2.9 n/a 4.3 4.4 4.5

MEDIAN 10.6 n/a 0.0 2.8 0.0

min 6.5 n/a 0.0 0.0 0.0

max 12.5 n/a 10.8 12.5 14.3

A

B n/a

C p = 0.012 n/a

D p = 0.022 n/a p > 0.050

E p = 0.006 n/a p > 0.050 p > 0.05

TOTAL VIRAL

SHEDDING

PAIRWISE

COMPARISON

T-TEST (Viral

shedding)

ANOVA p = 0.040

INFECTION RATES on July 25, 2020 by guesthttp://cvi.asm

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FLU-v GroupPLACEBO Group FLU-v Group

A/Swine/Iowa/15/30 A/Swine/Iowa/15/30

IFN-γ response IFN-γ response

PLACEBO Group

(fold-increase to -ve ctrol)

N = N =

(fold-increase to -ve ctrol)

7 8

VACCINATION VACCINATION

PRE- POST- PRE- POST-

7.0 10.3 3.4 5.2

1.3 3.2 1.3 1.1

13.0 12.1 31.8 33.5

min

max

MEDIAN

max

min

MEDIAN


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