MHRA Scientific Advice

Briefing Document

Proposed Vaccine: Flutcore - Universal Influenza Vaccine

Active Substance: VLPs based on Hepatitis B Core Antigen:

1. VLP1 (HA2.3,(M2e)3)

2. VLP2 (LAH3, K1)

Intended indication (s): Prevention of disease caused by influenza A virus

(The intention is to expand the vaccine and the indication to

include influenza B over the course of clinical development)

Sponsor: iQur Ltd

Consultancy company: ELC Group s.r.o.

Version: 01

Date: 9 November 2015

Contents

1. List of figures ................................................................................................ 1

2. List of tables ................................................................................................. 2

3. List of abbreviations ........................................................................................ 3

4. Summary ..................................................................................................... 4

Background information on the disease to be prevented .................................................................. 4

Influenza vaccines: current and future ..................................................................................... 5

Background information on the product ................................................................................... 6

5. Questions and company’s positions ....................................................................... 9

Questions on chemical, pharmaceutical and biological development .................................................... 9

Non-clinical development .................................................................................................. 25

Questions on toxico-pharmacological development ...................................................................... 26

Question on clinical development ......................................................................................... 34

Multidisciplinary question ................................................................................................. 41

6. List of references .......................................................................................... 42

1

1. List of figures

Figure 1: Structure and inserts of VLPs

Figure 2: Representation of VLP 1 and VLP 2 constituents

Figure 3: Description of manufacturing of MCB and WCB

Figure 4: Description of manufacturing process and process controls: USP

Figure 5: Description of manufacturing process and process controls: DSP

Figure 6: Manufacture of VLP1 and VLP2 drug products

Figure 7: Pre-infection chart

Figure 8: Survival chart (lethal challenge with H1N1)

Figure 9: Survival chart (lethal challenge with H3N2)

2

2. List of tables

Table 1: Specifications for VLP1 and VLP2 MCB and WCB

Table 2: Specifications for VLP1 and VLP2 drug substances

Table 3: Drug substance characterisation assays

Table 4: Specifications for VLP1 and VLP2 drug products

Table 5: Short term stability conditions: VLP1 and VLP2 drug substances at storage (-20°C)

and accelerated (2-8°C) temperatures

Table 6: Long-term stability conditions: VLP1 and VLP2 drug products stored at -20°C

Table 7: Intramuscular toxicity study outline

Table 8: Immunogenicity and H1 and H3 viral challenge studies in ferrets

Table 9: Clinical protocol synopsis

Table 10: Study event schedule

3

3. List of abbreviations

VLP Virus-like particle

HBc Hepatitis B core

MIR Major insertion region

HA Haemagglutinin

IFA, IFB Influenza A, B

LAH3 Single HA-stalk antigen which is specific to group 2 IFA viruses

HA2.3 Single HA-stalk antigen which is specific to group 1 IFA viruses

M2e Matrix 2 protein, integral in the viral envelope of the influenza A virus

cGMP Current good manufacturing practice

CMO Contract manufacturing organisation

RCB Research cell bank

MCBs Master cell banks

WCBs Working cell banks

YPD agar Yeast extract peptone dextrose

AOX Alcohol oxidase

PCR Polymerise chain reaction

qPCR Quantitative PCR

IPCs In-process controls

PPs Process parameters

SEC Size-exclusion chromatography

HRP Horseradish peroxidase

TMB Tetramethyl benzidine

GLP Good laboratory practice

pbl Peripheral blood lymphocytes

IMP Investigational medicinal product

MedDRA Medical Dictionary for Regulatory Activities

OD Optical density

TMP Transmembrane pressure

4

4. Summary

Background information on the disease to be prevented

Influenza is an infectious respiratory illness caused by infection with an influenza virus

presenting commonly with symptoms such as abrupt onset of fever, shivering, headache,

muscle ache and dry cough. More common complications of influenza are bronchitis and

pneumonia due to bacterial infections on top of the infection with the influenza virus. Rarer

but more severe complications are encephalitis (brain infections) and generalized infections.

These complications often require treatment in hospital and can be life threatening especially

in the very young, the elderly, and those in poor health.

Influenza viruses are one of the major infectious disease threats to the human population

owing to both the health impact of annual influenza and the tremendous global consequences

of influenza pandemics. Seasonal influenza alone causes 250,000 to 500,000 deaths and three

to five million cases of severe illness worldwide each year. A public health concern is that a

highly virulent influenza strain could lead to millions of deaths worldwide.

The influenza virus circulates worldwide, with a seasonal nature that is often hemisphere-

specific and mostly observed in temperate climates. Hence, existing methods of vaccine

selection, production and distribution may be best suited for seasonal nature of influenza;

however they are not well suited for preventing influenza in tropical countries where the virus

circulates year-round. Therefore these countries are mostly unprotected against seasonal

influenza and are particularly vulnerable to future pandemics.

Furthermore, the economic cost can also be very high as a severe epidemic may affect a

significant part of the working population, affecting productivity and overwhelming public

health systems. Anti-viral drugs – specific ones such as neuraminidase inhibitors oseltamivir

and zanamivir, or M2 inhibitors such as amantadine appear to have a limited benefit as they

need to be administered early in the infection and tend to provide only some reduction in the

duration and perhaps severity of symptoms of influenza. In addition, some strains of

influenza have developed anti-drug resistance. Hence, vaccination still remains the primary

approach for controlling influenza in persons and populations.

The current portfolio of subtype specific seasonal influenza vaccines do provide important

medical benefit, but their limitations have driven the field towards the “universal influenza”

vaccine concept that can overcome the virological phenomenon of antigenic drift and

antigenic shift. Moreover, the “universal influenza” vaccine is expected to ensure breadth of

protection against most strains and subtypes.

5

Influenza vaccines: current and future

Currently, the influenza vaccine needs to be updated annually, due to hypermutation of the

virus. Once the virus strains are predicted for the forthcoming influenza season, these are

usually prepared in hen eggs, a process which is both time consuming and costly. The

FLUTCORE vaccine is entirely recombinant and, because it uses invariant antigens, does not

require annual revision. Expression is carried out in yeast which is both rapid and relatively

cheap.

A further limitation of the annual seasonal influenza vaccine is that the product relies on an

accurate prediction of the impending virus strains. Failure to do this adequately results in an

ineffective vaccine. This was the case in the 2014-15 season in which up to 70% of H3N2

viruses in circulation were drift variants and thus not covered by the seasonal vaccine. Indeed

the Center for Disease Control rated the vaccine’s effectiveness as low as 23%.

The development of a universal influenza vaccine overcomes these limitations. Antigens are

chosen which are common to all influenza virus strains and so a viable vaccine can be made

regardless of annual mutation. A further advantage of this system is that the vaccine could be

stockpiled for use against future pandemics. Currently, no coherent strategy exists to combat

outbreaks similar to that seen in 2009. Although it is certainly possible to store known

pandemic strains, the rapid production of a vaccine to these is not currently feasible using egg

based technology within the timeframe of a pandemic. More importantly, it is much more

likely that the next pandemic will not be caused by a known virus but rather by an as yet

undetected variant. Therefore, stockpiling older pandemics is unlikely to be useful. However,

FLUTCORE does not suffer from these limitations and is directed against invariant antigens

which will almost certainly be present in any future pandemic strain.

6

Background information on the product

Introduction

iQur has developed a novel universal1 influenza vaccine, Flutcore, based on the tandem

core™ vaccine platform. This is a virus-like particle (VLP) composed of two Hepatitis B core

(HBc) proteins linked by a flexible linker sequence (figure 1). This construct increases VLP

stability and allows the presentation of multiple antigens in the VLP simultaneously. VLPs

are often chosen as an excellent vaccine vector since they are proteinaceous and do not carry

any risk of viral nucleic acid replication or recombination.

Figure 1: Structure and inserts of VLPs

The Tandem Core platform

The tandem core construct is a development of VLPs which form spontaneously when

multiple HBc antigens coalesce. Initially, two HBc molecules dimerise and then 90-120 of

these dimers, depending on symmetry, coalesce into a VLP. HBc molecules have a

characteristic “spike” structure which is comprised of two anti-parallel α-helices. At the tip of

this spike is the Major Insertion Region (MIR) into which third party antigens may be

inserted. Such VLP have been shown to be highly immunogenic and have the ability to

confer immunogenic properties to protein inserts within its structure.

However, wild-type HBc VLPs have a significant limitation; it has been found that large or

hydrophobic antigens make the HBc dimer unstable and thus prevent VLP formation

(Pumpens & Grens 2001). A failure to form a VLP leads to a significant loss of

1 Universal in this context means that the vaccine has been tested against a number of IFA strains but not in all known or potentially unknown strains

7

immunogenicity. The tandem core construct overcomes this limitation by physically linking

two HBc molecules in series. Furthermore, the system can now tolerate large or hydrophobic

antigens since the HBc dimer can no longer dissociate. As a further consequence of its

design, tandem core now has two MIRs capable of carrying multiple targets simultaneously. ,

thus making tandem core an ideal vaccine platform for large and multiple antigens.

Flutcore comprises two populations of VLPs (VLP1 and VLP2), both of which carry

antigens that are common to all strains of influenza A (figure 2)

VLP 1 (called pHe7HA2.3:M2e3): An antigen specific for the haemagglutinin (HA) stalk

region common to group 1 IFA viruses in MIR1 (major insertion region) as well as a triple

sequence from the M2e protein in MIR2. The triple M2e sequence has a consensus design

such that it will provide coverage against all known strains of IFA. The HA antigen is from

the conserved stalk region and has been designated HA2.3.

VLP 2 (called pHe7LAH3:e): A single HA-stalk antigen which is specific to group 2 IFA

viruses. Experimental evidence has shown that this antigen is exquisitely sensitive to steric

hindrance from antigens located in MIR2 and, is thus consequently only expressed in

isolation. This antigen is known as LAH3.

However, these antigens are also usually non-immunogenic unless they are expressed in the

context of a highly stimulating vector such as tandem core. Collectively, these targets are

proposed to provide protection from all known influenza A viruses. Currently, the

FLUTCORE project is designed only to protect against influenza A viruses. However, we are

actively investigating the possibility of developing another VLP containing antigens directed

against influenza B. There is considerably less variability within the IFB viruses so a

“universal” vaccine, based on tandem core technology, covering all clades should be

possible.

8

Figure 2: Representation of VLP 1 and VLP 2 constituents

The current stage of development has shown proof of the concept (immunogenicity and

protection) in mice using a non-GMP vaccine (for two individual VLPs with different protein

inserts, termed VLP1 and VLP 2) across several strains. The next stage is to transfer the

locked-down manufacturing process to the cGMP CMO in preparation for the production of

GMP lots for the pre-clinical package and ultimately the Phase 1 trial.

9

5. Questions and company’s positions

Questions on chemical, pharmaceutical and biological development

Question 1

Does the Agency concur with the proposed testing of the Master and Working Cell Banks?

Company’s position

The two elements of the vaccine are called PHe7HA2.3:M2e3 and pHe7LAH3:e and have

been given the designation of VLP1 and VLP2 respectively. Both constructs have been

cloned into pPICZc vectors and then used to transform the yeast Pichia pastoris (strain

KM71H). Both yeast clones were selected for high copy number expression and underwent

single cell cloning. Thus each RCB is derived from a single yeast clone. Clones for both

VLP1 and VLP2 were then fully characterised.

MCBs and WCBs of the vaccine were manufactured according to ICH Q5D guidelines

“Quality of Biotechnological/Biological Products: Derivation and Characterisation of Cell

Substrates Used for the Production of Biotechnological/Biological Products” and Ph. Eur.

general texts 5.2.3 (Cell Substrates for the Production of Vaccines for Human Use). These

cell banks were made to cGMP. Figure 3 below provides an overview on the establishment

of the MCB and WCB.

Figure 3: Description of manufacturing of MCB and WCB

Raw Material Unit Operation In Process Control

Thaw 1 vial RCB to prepare

MCB; OR 1 vial MCB to

prepare WCB

0.16 mL R/WCB; BMGY

medium

200mL medium in 1L

Erlenmeyer Flask.

Culture 250rpm, 30°C, 20 - 22

hours, until OD600 5

Culture density

OD600 = 5 for MCB

OD600 = 25 for WCB

80% Glycerol

HARVEST: Add ¼ volume of

80% glycerol (final 16%

vol:vol)

DISPENSE: 1mL into each of

200 cryovials

STORAGE

T = -80°C

The assays used for testing the MCB and WCB are tabulated below (table 1).

10

Table 1: Specifications for VLP1 and VLP2 MCB and WCB

Test Method Acceptance Criteria

Identity

Biochemical

Profiling

BioMerieux API 20C

AUX

Pichia pastoris

Sequence DNA sequencing of

tandem core plus

inserts

Identical to reference material

Morphology Light microscopy White, discreet colonies with

rounded form with entire

margins, raised elevation.

Growth

Characteristics

Growth on YPD agar

(yeast extract peptone

dextrose), 30°C for 3

days

Complies to OD limit

Purity Growth on antibiotic-

free plates

Only regular yeast colonies

detected

Copy number qPCR Comparable to historical data

and/or reference material

Viability Dilution plating and

counting

Report result

Summary of methods

Sequencing

The plasmid used to insert the tandem core sequence is such that it leads to integration

directly into the yeast genome by homologous recombination at the AOX locus. Furthermore,

once integration has been achieved, the AOX insertion site is regenerated allowing for further

recombination events to occur. Primers specific for the tandem core insertion are used to

amplify the region and then these PCR products are sequenced to ensure that full length

insertion has been achieved. However, this cannot provide an indication of the number of

insertions since the result is a consensus sequence.

Morphology

Phase contrast microscopy is used to determine the phenotype of colonies produced from

each yeast clone. The physical characteristics of both VLP have been determined and

colonies are compared to predefined reference standards.

11

Purity

Yeast are grown on agar plates devoid of all antibiotics and the presence of any contaminant

easily determined visually under phase contrast microscopy. The presence of any colony on

the agar plate which differs from the aforementioned reference standard will result in

rejection of the batch due to the presence of impurity.

Copy number

As mentioned previously, the plasmid used for transformation of the yeast leads to multiple

integration events. These integrations are identical and so sequencing is not able to determine

their number since each read is similar. To determine the number of integration events, we

have developed a bespoke quantitative PCR (qPCR) method in which primers specific for the

zeocin antibiotic resistance gene are used to amplify each insert and compared to a plasmid

standard. Standard curves are used to directly determine the number of inserted gene copies.

Question 2

Does the Agency concur with the In Process Controls (IPCs) and manufacturing process

parameters (PPs) identified for the manufacturing process for the drug substance for a Phase

1 clinical trial? Does the Agency have any other comments on the manufacturing process for

the drug substance?

Company’s position

Tandem core VLP are produced in yeast (Pichia pastoris) transformed with the integrating

plasmid pPICz. Expression is inducible by methanol, under the control of the AOX gene

found in the plasmid. VLPs are produced within the yeast cell and are not secreted. The

induced yeast must be lysed under high pressure before purification can begin. This lysate is

clarified by centrifugation and filtration prior to size exclusion chromatography (SEC). The

first SEC column (CL4B) uses a small bead size and VLP and other large material are

immediately captured in the void volume. The remaining 90% of other proteins are discarded.

The collected void volume is then applied to an S1000 SEC column which can then resolve

these large particles to isolate the VLP.

Figures 4 and 5 present the manufacturing process flow chart for the upstream and

downstream manufacturing process respectively (same for both VLP1 and VLP2).

12

Figure 4: Description of manufacturing process and process controls: USP

Raw Material Unit Operation In Process Control

Thaw 2 vials WCB

2X 1mL WCB; 2x 250mL

BMGY Culture medium in

2L flasks

Inoculum Expansion:

Temp = 30°C

Time = 16 – 18 Hrs

Agitation = 200rpm

Culture density:

Initial OD600 0.1-0.5

Final OD600 15-20

Pool 2 flasks to give approx.

500mL

Basal media

+ 4.35 mL/L of PTM salts

solution

Batch Phase initiated with

275mL inoculum (5%)

Initial Vol = 5.5L

Temp = 30°C

pH = 5.0 ± 0.25

Agitation 400-1200rpm

Air flow rate 0.5vvm

Minimum DOT 30%

Time = 18 – 20 hrs

End criteria: O2 Spike

(controlled by CO2)

Initial OD600 = 0.9 – 1.5

Measure every 2-3 hrs

Spike OD600 = 110 - 140

Induction Media:

Feed with

Glycerol/Water/MeOH/PTM

salts

Fed Batch Induction Phase

Feed rate 2.5 – 5.0 mL/h/L

initial volume

End criteria: 48 hrs after

induction

Measure every 4 hrs

Final OD600 = 400 -500

Harvest by centrifugation (10

mins @ 5,000 g) discard

supernatant, store pellet

(160g/L)

Biomass weight

STORAGE

For time ≤ 30 days, T = -20°C

For time > 30 days, T = -80°C

13

Figure 5: Description of manufacturing process and process controls: DSP

Lysis Buffer:

50mM MOPS

5mM DTT

2mM AEBSF protease

inhibitors

5u/mL benzonase

Adjust pH to 7.5 with 3M

NaOH

Thaw 25g Biomass and disrupt

by Micronisation:

Resuspend in buffer to 5% w/v

Lysis by 3 passes at 500Bar

Temp = 2-8°C

Solubilisation Buffer:

10% v/v Triton X-100

Add 10mL/L

Solubilisation:

Triton X-100 (to 0.1% v/v final

concentration)

Time = 1 hr

Temp = 4°C

Clarification:

Centrifuge @ 20,000g

Time = 30 min

Temp = 4°C

Discard Pellet; Process

Supernatant (c. 450mL)

Dilution Buffer

20mM Tris

10mM EDTA

2M Urea

Dilution and buffer adjustment

Add equal volume dilution

buffer (c.900mL)

Protein concentration (total

protein)

Depth filtration:

0.8µm/0.45 µm /0.2 µm

TFF Hollow fibre filter

150cm2

D02-E750-05-N

Spectrum Labs

TFF concentration 30-40X:

750kDa cut-off

TMP = 6.0psi

Flux permeate 30LMH

Filtration:

0.2µm/0.1µm

Homogenate intermediate

20 – 30 mL

Appearance, pH,

Total protein

AKTA Sepharose CL-4B

2.6 x 92cm

Column Buffer:

20mM Tris, pH 8.4

5mM EDTA

1M Urea

SE chromatography 1:

Flow rate: 22cm/h (2mL/min)

Temp = room temp

Collect void volume peak

14

AKTA Sephacryl S-1000

5 x 92 cm

Column Buffer:

20mM Tris, pH 8.4

5mM EDTA

SE chromatography 2:

Flow rate: 15cm/h (5mL/min)

Temp = room temp

Fractions held at RT < 10hrs.

Then kept at 4°C 48hrs

Individual fractions SDS-

PAGE + W Blot

Fractions chosen based on

Ag stain and densitometry

Pool fractions which meet pre-

set criteria of purity and

concentration

Buffer:

20mM Tris, pH 8.4

5mM EDTA

TFF concentration:

Increase concentration to 0.1 –

0.4 mg/mL

750kDa cut-off

TMP = 3.0 psi

Temp = 4°C

Final Filtration:

0.2µm

Drug Substance

container-closure Fill into drug substance

container-closure Test according to

specifications

STORAGE -20°C

15

Question 3

Does the agency concur with the content and completeness of the specification and testing

methodology for the drug substance for a Phase 1 clinical trial?

Company’s position

The Drug Substance specifications are tabulated below (Table 2). The specifications have

been established according to ICH Q6B. It is therefore the Company’s position that the

analytical methods proposed for the Drug Substance specification are sufficient to ensure the

quality of the Drug Substance for use in a Phase 1 clinical trial.

Table 2: Specifications for VLP1 and VLP2 drug substances

TEST Method Acceptance criteria

pH pH (Ph. Eur. 2.2.3)

8.4 +/- 0.5

Identity

SDS-PAGE and Western

Blot

Major band at 58kDa for

VLP1 and 48kDa for VLP2

reacts with specific antibodies

for HBc, HA2.3, LAH3 and

M2e

Purity

SE-HPLC Peak elution volume,

homogenous with no shoulders

SDS-PAGE, Coomassie

Blue

>75% core related protein

Assay Protein concentration (total

protein)

Report result

Impurities

P. pastoris HCP ELISA

Report result

HC DNA qPCR / picogreen

< 10 ng / dose

Safety

Endotoxins (Ph. Eur. D

2.6.14)

< 50 IU/mg

TYMC/TAMC Ph. Eur.

2.6.12

<10 cfu / 100 mL

Antigenic activity In vitro assays under

development*

Comparable to reference

standard

* by ELISA or SPR

Characterisation assays, additionally employed for testing of drug substance and reference

standards, are described in Table 3 below.

16

Table 3: Drug substance characterisation assays

Test Method

Protein identity Western blotting

Protein purity SDS-PAGE

VLP formation HPLC-SEC

VLP morphology Electron microscopy

A brief overview of the drug substance and characterisation analytical methods is given

below.

Non-compendial methods:

SDS-PAGE (reduced): identity and purity

Proteins are separated according to size by electrophoresis and visualised by staining with

Coomassie Blue. Sizes are estimated by comparison with known standards. Identity of

samples by comparison with reference standard.

Western blot: identity

Proteins separated by SDS-PAGE are blotted onto a membrane and visualised by specific

staining with an antibody specific for the target antigen.

SE-HPLC: purity

Sample is run on a calibrated analytical size exclusion column and the position and

homogeneity of the product peak compared to a reference standard. This technique confirms

the structural integrity of the assembled VLPs.

Electron microscopy: purity

The homogeneity and correct size class of the assembled VLPs is visualised directly.

Protein concentration: wuantity

The total protein content of samples is determined by a colorimetric assay according to kit

manufacturer’s instructions.

HCP ELISA:

The Cygnus Technologies Pichia pastoris assay is a two-site immunoenzymetric assay used

to quantitate host cell protein. Samples containing Pichia pastoris HCPs are reacted in

microtiter strips coated with an affinity purified capture antibody. A second horseradish

peroxidase (HRP) enzyme labelled anti-Pichia pastoris antibody is reacted simultaneously

resulting in the formation of a sandwich complex of solid phase antibody-HCP-enzyme

labelled antibody. The microtiter strips are washed to remove any unbound reactants. The

substrate, tetramethyl benzidine (TMB) is then reacted. The amount of hydrolysed substrate

is read on a microtiter plate reader and is directly proportional to the concentration of Pichia

pastoris HCPs present.

17

HC DNA:

The presence of contaminating host cell DNA is firstly prepared using the PreSeq® Residual

DNA kit (Life Technologies). This also includes relevant positive and negative controls. The

amount of DNA is measured using the resDNASEQ® Quantitative Pichia pastoris DNA Kit

(Thermo-Fisher) which is a quantitative PCR (qPCR)-based system for the detection of host

cell DNA from Pichia pastoris cells. The kit is reliable and rapid with a sensitivity as low as

15pg DNA/mL test sample. This performance helps ensure a high degree of confidence in

quantitation data obtained from a broad range of sample types, including in-process samples

and to final product. Detection is regardless of whether the sample contains high molecular

weight or sheared DNA.

Question 4

For the planned Phase 1 study, the final drug product presentation is proposed to be

composed of separate vials of VLPs, adjuvant and dilution buffer in order to provide

flexibility in trial design where dose escalation and formulations both with and without

adjuvant are required. The final dosage form will be assembled at the clinical site as soon as

practical before administration. Does the Agency concur with this plan and can they advise

on appropriate QC/QA procedures required at the clinical site?

Company’s position

The proposed Phase 1 trial will be the first exposure of human subjects to the vaccine and a

primary objective is to evaluate a number of dose levels and doses with and without the

addition of Alhydrogel adjuvant to select an appropriate formulation for further development.

In the proposed approach the drug product is manufactured as four separate sterile

components (two separate drug substances, dilution buffer and Alhydrogel suspension) and

provided to the clinical site, where individual dosage forms are assembled in a clinical

pharmacy to produce a variety of vaccine dose levels and formulations including placebos.

The assembly process will be tested by performing qualification runs to verify that the quality

of the assembled dosage forms is acceptable, including a 24hr in-use stability study. This

approach is considered to be the most efficient and flexible manner in which to achieve the

objectives of the Phase 1 trial.

18

Question 5

Does the Agency concur on the IPCs identified for the manufacturing process for the DRUG

PRODUCT for a Phase 1 clinical trial? Does the Agency have any other comments on the

manufacturing process for the drug product?

Company’s position

The Drug Product will be manufactured as two separate components:

a) A solution of VLP1 filled aseptically into suitable container closures after sterile

filtration and stored at -20°C

b) A solution of VLP2 filled aseptically into suitable container closures after sterile

filtration and stored at -20°C

Additionally, two further components will be required, consisting of:

c) A dilution buffer filled into suitable container-closure and sterilised by autoclaving

and stored at 2-8°C

d) Alhydrogel suspension filled into suitable container-closure and sterilised by

autoclaving and stored at -20°C

The choice of the final formulation and dilution buffer for the VLP drug product is currently

being finalised. It will be a conventional buffer used for parenteral administration of

vaccines. Alhydrogel is commercially purchased. It is repackaged into quantities appropriate

for the clinical administration schedule.

Figure 6 describes the manufacturing process for VLP1 and VLP2 drug products.

Figure 6: Manufacture of VLP1 and VLP2 drug products

Raw Material Unit Operation In Process Control

Thaw VLP Drug Substance

Bulk

Identity: SDS-PAGE, W-Blot

Formulation Buffer

Dilute to appropriate

concentration

Protein concentration (total

protein)

Sterile Filtration

Aseptically fill by volume into

Drug Product containers

STORAGE

T = -20°C

Test according to

specifications

The non-compendial IPC analytical methods applied to the Drug Products are as already described

under questions 1 and 3 of the Drug Substance above.

19

Question 6

Does the agency concur on the content and completeness of the specification and testing

methodology for drug product final containers for a Phase 1 clinical trial?

Company’s position

The Drug Product specifications for VLP1 and VLP2 are tabulated below (Table 4). The

specifications have been established according to ICH Q6B. It is therefore the Company’s

position that the analytical methods proposed for the Drug Substance specification are

sufficient to ensure the quality of the Drug Substance for use in a Phase 1 clinical trial.

Table 4: Specifications for VLP1 and VLP2 drug products

TEST Method Acceptance criteria

Appearance

Clarity (Ph. Eur.

2.2.1)

Clear solution

Colour (Ph. Eur.

2.2.2)

Colourless to slightly yellow

Visible particles Visual inspection (Ph.

Eur. 2.9.20

Practically free from visible particles

pH pH (Ph. Eur. 2.2.3) 8.4 +/- 0.5

Osmolality Ionic strength To be determined prior to CTA

Identity

SDS-PAGE and

Western Blot

Major band at 58kDa for VLP1 and

48kDa for VLP2 reacts with specific

antibodies for HBc, HA2.3, LAH3

and M2e

Purity

SE-HPLC Peak elution volume, homogenous

with no shoulders

SDS-PAGE,

Coomassie Blue and

quantitative scan

>75% core protein

Assay Protein concentration

(total protein)

Limits to be determined based on

safety data*

Safety

Endotoxins (Ph. Eur.

D 2.6.14)

< 50 IU/mg

Sterility; membrane

filtration (Ph. Eur.

2.6.1)

Sterile

Antigenic activity

(or in vivo potency

on an adjuvant

reconstituted

product)

In vitro assays under

development**

or in vivo

immunogenicity

Report result

Extractable volume Ph. Eur. 5.6 Minimum 0.5mL

* Limits to be determined based on maximum dose to be used in Phase 1 trial and supported

by pre-clinical safety data - currently expected to be at nominal 600ug/mL

20

** By ELISA or SPR

The non-compendial analytical methods applied to the Drug Products are as already

described under questions 1 and 3 of the Drug Substance above.

Potency:

Initial potency testing will be based on quantitative immunogenicity testing in mice. End-

point titrations of antibodies specific for each element of the vaccine will be used to set a

baseline. These antibodies will have been generated from reference quality VLP material and

thus will be representative of the final product. As development of analytical techniques

progresses, alternative in vitro methods will be correlated with the in vivo data with the aim

of developing a release test based only on in vitro data.

Data has shown that strong expression of target antigens on a VLP correlates with in vivo

seroconversion. Therefore, a measurement of antigen expression is a reasonable surrogate for

biological activity. Conventionally, western blots are problematic for antigen screening since

the technique relies on denaturing the protein during electrophoresis. Antibodies which are

active in western blot are available for the FLUTCORE vaccine and so it is proposed to test

antigen expression as follows;

(a) Core protein: Antibodies to the N-terminus of core protein (10E11) and the C-

terminus (14E11) are used routinely.

(b) M2 antigen: Can be detected using the 14C12 monoclonal

(c) HA antigens: iQur is currently developing bespoke monoclonals to HA2.3 and LAH3.

However, anti-sera made to these antigens have already been shown to be active in

western blot.

Although western blotting is capable of detecting the presence of antigens, it is not a fully

quantitative technique. Therefore, iQur has developed a plasmon resonance based technique

using BIAcore. A method has been developed in which VLP are immobilised to a gold

BIAcore chip using an antibody. Antibodies specific for each vaccine insert can then be

passed over this complex and their binding fully quantitated. Thus, a reliable measure of

vaccine quality and stability may be collected. It is planned to test the in vitro potency

method (on non-adjuvanted VLP1 and VLP2) head-to-head with the mouse in vivo potency

assay (using adjuvanted VLP1 and adjuvanted VLP2 in final reconstituted co-mix) to

establish equivalence after forced degradation and spiking studies.

Thus, we believe that antigen expression can be monitored on the surface of VLP over time

and that this is a viable method to assess vaccine stability in lieu of the in vivo

immunogenicity test in the stability studies.

21

Question 7

Does the Agency concur with the proposed stability strategy to support the use of the Drug

Substance and Drug Product for a Phase 1 clinical trial?

Company’s position

The stability of the VLP Drug Substances (table 5) and Drug Products (table 6) will be

assessed in controlled studies as described below.

At the time of submission of the CTA iQUR expects to have supportive stability data

available for the non-GMP engineering batch as follows:

3 months at long-term storage temperature (-20°C)

3 months accelerated (2-8°C)

The stability of the GMP batch to be used in the clinical Phase 1 study will be monitored

concurrently during the period of the trial.

iQUR proposes that the clinical trial batch (VLP1 and VLP 2 drug products) be assigned a

shelf-life of 6 months.

22

Table 5: Short term stability conditions: VLP1 and VLP2 Drug Substances at storage (-20°C) and accelerated (2-8°C) temperatures

Test parameter Acceptance criteria Results

Initial 1M 3M 6M 9 M 12 M

2-8°C -20°C 2-8°C -20°C 2-8°C -20°C -20°C -20°C

Appearance Clear and colourless

solution

X X X X X X X X X

pH 8.4 +/- 0.5 X X X X X X X X X

Purity

SDS-

PAGE/Western

blot

>75% core protein X X X X X X X X X

Purity

SE-HPLC

Homogeneous major

peak

X X X X X X X X X

Protein

Concentration

(total protein)

TBD X X X X X X X X X

In vitro potency Report Result X X X X nd X nd X nd

X =Test point scheduled

nd = Not determined

23

Table 6: Long-term stability conditions: VLP1 and VLP2 drug products stored at -20°C

Test Parameter Acceptance Criteria Results

Initial 3 Months 6 Months 9 Months 12 Months

Appearance Clear and colourless solution X X X X X

pH 8.4 +/- 0.5 X X X X X

Purity

SDS-PAGE

>75% core protein X X X X X

Purity

SE-HPLC

Homogeneous major peak X X X X X

Protein

Concentration

(total protein)

TBD* X X X X X

Bioburden

TYMC

Report Result X X X X X

Bioburden

TAMC

Report Result X X X X X

In vivo potency

on adjuvanted

product

Report Result X nd nd nd X

In vitro potency Report Result X X X X X

* Limits to be determined based on maximum dose to be used in Phase 1 trial and supported by pre-clinical safety data - currently expected to be

at nominal 600ug/mL

X = Test point scheduled

nd = Not determined

24

In-use (reconstituted) stability: assembled drug product

As described elsewhere the final dosage forms will be assembled as close to the time of

administration as practical in the pharmacy of the clinical site. In order to demonstrate that

the assembled product remains stable for use the same day a short-term 24 hour in-use

stability program will be performed on material taken from the engineering batches at both

2-8°C and room temperature. As the reconstituted product is Alhydrogel adjuvanted, this

limits the number of tests that are possible due to the strong interaction between the

antigens and adjuvant.

The stability indicating assays to be used in the reconstituted study will be:

Proportion of VLPs bound to AlOH (total protein in solution by Bradford Assay,

identification of VLP1 vs VLP2 by SDS-PAGE and W-blot)

In vivo potency at T0 and T24

25

Non-clinical development

Since this is a novel “universal influenza” vaccine, the proposed nonclinical development

programme includes toxicology testing and primary pharmacodynamic study, in line with

the “Guideline on influenza vaccines Non-clinical and clinical module

EMA/CHMP/VWP/457259/2014.” Further according to this guidance document,

considering this is a vaccine product, dedicated safety pharmacology studies,

pharmacokinetic studies, genotoxicity and carcinogenicity studies will not be conducted for

this vaccine. The local tolerance assessment will be evaluated as a part of proposed toxicity

study.

26

Questions on toxico-pharmacological development

Question 8

Does the agency accept the study design for toxicity study including chosen animal model?

Company’s position

In line with the “Guideline on influenza vaccines Non-clinical and clinical module

EMA/CHMP/VWP/457259/2014”, the nonclinical safety studies should be conducted in

compliance with Good Laboratory Practice (GLP). Further, the studies investigating the

toxicological effects of the candidate vaccine can be performed in one animal species of

relevance (e.g. rats, ferrets, rabbits).

Accordingly, the company intends to conduct GLP-compliant Intramuscular Toxicity Study

with the candidate vaccine in rats. The study outline is as follows:

Table 7: Intramuscular toxicity study outline

Test substance: Flu Vaccine

Duration: 4 weeks followed by a 2 week recovery period. Main study animals

to be terminated on Day 35 and recovery animals on Day 42.

Frequency of

dosing

Three doses of the study dose will be administered on Days 0, 14

and 28 (0.5mL).

Route: Intramuscular

Species: Rats (Sprague Dawley)

Age at start of

treatment:

6-7 weeks (>220g)

Groups: 1 2

Treatment: Control Highest dose proposed for the

human study

Animals - main: 10M + 10F 10M + 10F

27

Serial Observations:

Occasions (Week) Details

Bodyweights: Week -1, Day 0, 14, 28, 35

and 42

Food consumption: Week -1 until termination Weekly

Clinical observation: Twice daily

Daily

Pre-dose and daily after

each dose for seven days

Mortality check

Post-dose observations

Local irritation

Ophthalmoscopy: Pre-treatment

Days 14, 28 and 42

All study animals and spares

All study animals

Standard observations using a binocular

indirect ophthalmoscope

Haematology: Days 7, 14, 21, 28, 35 &

Termination

First 5 animals/sex/group and recovery

animals

red blood cell (erythrocyte) count,

haemoglobin, haematocrit, mean

corpuscular volume, mean corpuscular

haemoglobin, mean corpuscular

haemoglobin concentration, platelet count,

white blood cell (leukocyte) count,

differential blood cell count, blood smear,

reticulocyte count, Prothrombin time,

activated partial thromboplastin time,

fibrinogen

Blood chemistry Days 7, 14, 21, 28, 35 &

Termination

Second 5 animals/sex/group and recovery

animals

Glucose, urea nitrogen, creatinine, total

protein, albumin, globulin,

albumin/globulin ratio, cholesterol, total

bilirubin, alanine aminotransferase, alkaline

phosphatase, gamma glutamyltransferase,

aspartate aminotransferase, calcium,

inorganic phosphorus, sodium, potassium,

chloride, triglycerides

Immunogenicity Predose and Day 35 Analysis of M2e, LAH1 and LAH3

antibodies by ELISA

Necropsy and Organ

Weights:

Day 35

Day 42

All main study animals

All recovery animals

47 tissues to be retained and processed.

28

Histopathology: All study animals

47 tissues to be examined (including sites

of injection)

Question 9

a. Does the agency accept the overall study design for immunogenicity testing

including chosen animal model and chosen strains to address breadth of protection?

b. To encompass animal welfare in the proposed immunogenicity testing, a moderate

to severe disease will be considered with a non-lethal dose for ferrets, in line with

the guidance EMA/CHMP/VWP/457259/2014. On ethical grounds, and that with

the lethal dose, animals deteriorate very quickly, non-lethal dose is justifiable for

viral challenge in ferrets in order to have comprehensive assessment of all

“important” immunogenicity endpoints. Is this acceptable to the agency?

Company’s position

Vaccine development has, to date, been carried out in the mouse model. This is because it

allowed for rapid screening of vaccine candidates and also full protection from lethal

challenge could be demonstrated. Many strains of influenza are not lethal in alternative

models.

In the example shown below, mice have been immunised three times, with weekly interval,

using the FLUTCORE vaccine comprising of two VLP. Sera were taken at day 28 and

tested on recombinant haemagglutinin molecules or M2e peptides using an ELISA. The

graph shows that seroconversion has been achieved to both H1 and H3 influenza serotypes,

as well as to the normally non-immunogenic, M2e target.

29

Figure 7: Pre-infection chart

These immunised animals were then split into two groups prior to lethal influenza

challenge. Group 1 received x5 LD50 of H1N1 (PR8). Vaccinated (H1v) animals showed

100% protection, whilst all control (H1c) animals died.

30

Figure 8: Survival chart (lethal challenge with H1N1)

Similarly, Group 2 animals were challenge with x3 LD50 of H3N2 (X31) and, again,

vaccinated (H3v) animals survived whilst the majority of control animals (H3c) perished.

31

Figure 9: Survival chart (lethal challenge with H3N2)

In line with the “Guideline on influenza vaccines Non-clinical and clinical module

EMA/CHMP/VWP/457259/2014”, primary pharmacodynamic study is planned in ferrets to

study the protective efficacy of the candidate vaccine against flu virus challenge. In order to

address the universality of the vaccine protection, separate challenge studies are planned

against diverse strains such as H1N1 and H3N2.

According the guidance EMA/CHMP/VWP/457259/2014, ferrets have been chosen as the

animal models for these challenge studies because the disease pathogenesis, clinical signs

and mechanisms of immunity closely resemble human disease. The animals easily succumb

to a wide range of human influenza viral strains and display all of the classic symptoms

associated with a flu infections (very similar to what one would see in humans). Moreover,

the published literature as enclosed in the below list of references confirm ferrets as the

preferred model for immunogenicity testing 5-10.

32

The study outlines are as follows:

Table 8: Immunogenicity and H1 and H3 Viral Challenge studies in Ferrets

Purpose: To assess the effects of a vaccine on Influenza in ferrets

Route: Vaccine (i.m.), virus (i.n.) H1 or H3

Species: Ferret (male)

Groups: 1 2 3 4

Treatment: Positive control Lead Candidate with

adjuvant

(PHe7HA2.3:M2e3

pHe7LAH3:e)

Lead Candidate without

adjuvant

(PHe7HA2.3:M2e3

pHe7LAH3:e)

Negative

control

(adjuvant

Alhydrogel

suspended in

the buffer)

Vaccine Dosing: Days 0, 14 and

28

Days 0, 14 and 28 Days 0, 14 and 28 Days 0, 14 and

28

Dose volume

and route

0.2 mL, IM (0.1 mL per site, 2

sites)

0.2 mL, IM (0.1

mL per site, 2 sites)

0.2 mL, IM (0.1

mL per site, 2 sites)

0.2 mL, IM (0.1

mL per site, 2

sites)

Intranasal

challenge

with virus

Day 35 (but based on

immunogenicity

results)

Day 35

(but based on

immunogenicity

results)

Day 35

(but based on

immunogenicity

results)

Day 35

(but based on

immunogenicity

results)

Animals: 6M 6M 6M 6M

33

Experimental procedures:

Vaccination

Animals will be vaccinated (intramuscularly) with negative control or Test/Positive control vaccines

on days 0, 14 and 28.

Infection

Animals will be infected intranasally with either an infectious dose of the H1N1 or H3N2 influenza

strain 7 days after the seroconversion is achieved. Infection and other parameters will then be

assessed for next 6 days.

Blood sampling

Blood samples will be taken via a suitable vein on days 0 (prior to dosing), 7, 14, 21, 28 and 35. Sera

will be used for analysis of M2e, LAH1 and LAH3 antibodies by ELISA.

End point analysis

Nasal swabs (for measurement of viral titre) will be taken on days -1, 2, 4 and 6 (where Day 0

would be the day of infective challenge) measured by TCID50 assay. Once seroconversion is

achieved on Day 28, these days would correspond to study days 34, 37, 39 and 41.

Blood sampling (for analysis of M2e, LAH1 and LAH3 antibodies by ELISA) will be taken on days

0 and 6 (where Day 0 would be the day of infective challenge). Seroconversion is expected by Day

28 and thus these days would correspond to study days 35 and 41.

Spleens will be collected at termination for IFN-γ ELISpots.

Clinical observations

Bodyweights, body temperature (via an implanted chip) and activity and symptom scores will be

taken daily (days -3,-2, -1, 0, 1-6 inclusive, where Day 0 would be the day of infective challenge).

Again, assuming seroconversion occurs by Day 28, these days would correspond to study days 32-

41.

Termination

On 6th day after infective challenge, after the final samples or observations have taken place, the

ferrets will be killed by an approved method. Lung will be removed aseptically and stored in

transport medium for transport to a third party laboratory for viral load determination.

Analysis

Data analysis: Statistical analysis, to compare viral titre in nasal swabs in the treated and non-treated

groups will be performed using an appropriate statistical test.

34

Question on clinical development

Question 10

Does the agency concur with the proposed Phase I study design for the candidate vaccine,

including study objective, eligibility criteria and study endpoints?

Company’s position

The proposed clinical development programme comprises of pre-licensure vaccine clinical

trials in three phases.

- Phase I study to assess safety and immunogenicity of the candidate vaccine with

study outline planned as below.

- Phase II study to further evaluate the safety and immunogenicity of the product and

to determine the optimal vaccine dose, and dosing schedule.

- Phase III study to provide critical documentation of effectiveness and important

additional safety data required for licensing.

35

Phase I study outline Table 9: Clinical protocol synopsis

Name of Sponsor/Company :

iQUR Ltd

Name of Finished Product :

Flutcore

Name of Active Ingredient:

VLPs based on Hepatitis B Core Antigen: VLP1(HA2.3,(M2e)3); VLP2(LAH3, K1)

Title of the study :

A Phase I, randomized, double-blind, placebo-controlled dose escalating study to evaluate the

safety, reactogenicity and immunogenicity of the novel universal influenza A virus (IAV) vaccine

based on the tandem core vaccine platform compared with placebo in healthy adult volunteers

Investigator :

To be decided

Study Centre :

To be decided

Studied period (years) :

8 months

Phase of development:

Phase 1

Objectives :

1) To evaluate the safety, tolerability and immunogenicity of ascending doses of the candidate

vaccine.

2) To explore the dose response to the vaccine.

3) To explore the necessity of including the adjuvant Alhydrogel.

4) To demonstrate the potential for the “Tandem Core” platform to safely generate significant

immune responses to heterologous antigens.

Endpoints:

Primary Safety:

Proportion of subjects experiencing Serious Adverse Events possibly associated with vaccination;

Secondary Safety:

Proportion of subjects experiencing any Adverse Events possibly associated with vaccination.

Primary Immunogenicity:

Number of subjects experiencing at least a 2.5-fold increase in systemic IgG to flu epitopes (M2e,

LAH2.3 and LAH3) carried by the vaccine. These data will be expressed as absolute titres and

geometric means.

Secondary Immunogenicity:

Magnitude of antibody and cell-mediated immune responses to influenza epitopes (geometric mean

titres and fold-increase in titres in serum, cellular responses will be assessed by restimulating frozen

36

peripheral blood lymphocytes (pbl) with specific influenza antigens. Stimulated pbl will be assessed

for proliferation, activation and cytokine secretion.

Methodology :

This is a single-centre, double-blind, randomised, placebo-controlled, Phase 1 trial to evaluate the

safety, tolerability and immunogenicity of the Flutcore vaccine in healthy adult volunteers.

The study will evaluate three escalating dose levels of the vaccine (X/5, X, 3X) which will be

defined based on pre-clinical results before submission of the CTA. All subjects will receive three

doses of vaccine or placebo 28 days apart. Vaccine and placebo will be administered as i.m

injection and subjects monitored for at least 4 hours after dosing before discharge. They will be

actively monitored for safety in the four weeks following each dose and followed up at 6 months

after the first dose to monitor any emergent serious or chronic health issues.

Initial tolerability of the lowest dose versus a placebo control will be evaluated in cohort 1

consisting of approximately 10 subjects, randomised 8:2 to receive (low dose vaccine plus

adjuvant): (placebo):

Cohort 1: Three doses of X/5 :AlOH administered on Day 0, Day 28 and Day 56.

If the first dose of vaccine is well tolerated, a new cohort 2 will be recruited consisting of

approximately 10 subjects, randomised 8:2 to receive (mid dose vaccine plus adjuvant): (placebo)

Cohort 2: Three doses of X:AlOH on Day 0, Day 28 and Day 56.

If the first dose of vaccine is well tolerated, a new cohorts 3 will be recruited consisting of

approximately 10 subjects, randomised 8:2 to receive (high dose vaccine plus adjuvant): (placebo)

Cohort 3: Three doses of 3X:AlOH administered on Day 0, Day 28 and Day 56.

If the first dose of vaccine is well tolerated, a new cohorts 4 will be recruited consisting of

approximately 10 subjects, randomised 8:2 to receive (high dose vaccine plus adjuvant): (placebo).

If there are concerns about the tolerability of the vaccine in Cohort 3, then the new cohort 4 will

instead receive the mid dose vaccine without adjuvant.

Cohort 4: Three doses of X, or 3X without AlOH administered on Day 0, Day 28 and Day

56.

For cohort 1 only a sentinel group consisting of one active and one placebo vaccine will be dosed a

minimum of 24 hours before the rest of the cohort. Only if no clinically significant AEs are

observed in these subjects will the rest of the cohort be dosed.

Within a cohort, the second and third doses will only be given if safety and tolerability of the

previous dose is acceptable. This assessment will be based on evaluation of blinded data up to 7

days after each dose by the PI and medical monitor.

Cohort 2 will be administered their first dose at least 14 days after the first dose of cohort 1,

37

assuming that the latter is well tolerated based on blinded data to Day 7. Cohorts 3 and 4 will be

administered their first dose at least 14 days after the first dose of the preceding cohort, assuming

that the latter is well tolerated based on blinded data to Day 7.

The timing and frequency of assessments are outlined in the Study Event Schedule.

Study halting rules

The study will be halted, and all safety data assessed, prior to a decision on recommencing if any of

the following occur:

Any serious adverse event possibly related to vaccination

Any of the following AE’s graded as moderate or severe occurring in 3 or more subjects in

a cohort: Fever, myalgia, local site reaction

Any other reason of medical concern

For the purpose of this assessment severe fever is defined as ≥39.0°C and moderate fever as 38.5°C

– 38.9°C in two measurements taken at least 30 minutes apart. Other AE’s are regarded as moderate

if they interfere with normal activity and are only partially relieved with symptomatic treatment or

severe if they reduce or prevent normal daily activity and are not relieved with symptomatic

treatment.

During this safety data assessment period, which may require unblinding of safety data as deemed

necessary, no new subjects will be vaccinated. Ongoing subjects will receive their second or third

vaccination at the discretion of the investigator and medical monitor.

The entire study will be halted if any subject experiences a SAE possibly attributable to the

vaccine.

The safety data assessment will be carried out by the principal investigator, independent medical

monitor, and sponsor representative who will be jointly responsible for deciding whether or not to

re-start the study. An outline of the extent of the safety assessment conducted and justification for

the decision taken will be documented in the study file.

Number of subjects (planned) :

Approximately 40 subjects will be randomised for this study;

It is considered that 40 subjects (8 low dose vaccine: 8 medium dose vaccine: 8 high dose vaccine:

8 Medium or high dose without adjuvant: 8 placebo) would be appropriate for allowing a

meaningful measure of the frequency of AEs in the active versus placebo groups and estimating the

frequency of immune responses to key antigens.

In order to maximize the chance that a full cohort is enrolled, alternate subjects (3 per cohort) will

be invited to attend for the first vaccination day. There is no intention to replace missing or drop-out

subjects.

Main criteria for inclusion :

1. Male or female age ≥18 and ≤50 years.

2. General good health, without significant medical illness, physical examination findings or

clinical laboratory abnormalities.

3. Negative serum pregnancy test at screening and a negative urine pregnancy test before

38

immunisation for female subjects of childbearing potential. Females of childbearing potential

must not be breastfeeding and must agree to use an efficacious hormonal or barrier method of

birth control during the study. Abstinence is acceptable. Female subjects unable to bear

children must have this documented (e.g. tubal ligation or hysterectomy) or must have negative

pregnancy tests.

4. Willingness to participate in the study after all aspects of the protocol have been explained and

written informed consent obtained.

5. Completion of a training session and demonstrated comprehension of the protocol procedures.

6. Availability for the study duration, including all planned follow-up visits.

Exclusion criteria at screening:

General health criteria

1. Acute or chronic, clinically significant pulmonary, endocrine, autoimmune, neurological,

cardiovascular, psychiatric, metabolic, hepatic or renal functional abnormality, as determined

by medical history, and physical examination tests, which in the opinion of the investigator,

might interfere with the study objectives. Some medical conditions which are adequately

treated and stable would not preclude entry into the study. These conditions might include

stable asthma controlled with inhalers or mild hypertension stably controlled with a single

agent.

2. Immunodeficiency, malignancy or receiving immunosuppressive therapy

3. Previous adverse reaction to vaccination

4. History of allergy to yeast or yeast extract

5. Significant abnormalities in screening hematology, serum chemistry, urinalysis or EKG, as

determined by PI or PI in consultation with the medical monitor and sponsor.

6. Presence in the serum of HIV antibody, HBsAg, HBcAb, or HCV antibody.

7. Subjects who have significant scarring, tattoos, abrasions, cuts or infections at the dose site that

in the opinion of the Investigator could interfere with evaluation of injection site local reactions

8. Evidence of current excessive alcohol consumption or drug dependence.

9. Recent vaccination or systemic infection (within the 4 weeks before vaccination).

10. Subjects who have received a flu vaccine in the last 12 months or who anticipate receiving it

within the duration of the study including follow up.

11. Receipt of an investigational drug within 30 days of the initial vaccine/placebo dose for this

study.

12. Any other criteria which, in the investigator’s opinion, would compromise the ability of the

subject to participate in the study, the safety of the study, or the results of the study.

13. Pregnancy, risk of pregnancy, or lactation.

14. Use of any medication known to affect the immune function (e.g., corticosteroids and others)

within 30 days preceding the first vaccination or planned use during the active study period.

39

Concomitant medication(s):

Only concomitant medications approved by the study physician will be used during the study.

Subjects taking regular medication (i.e., birth control pills) prior to enrolment will be allowed to

continue unless it is specifically excluded as part of the inclusion/exclusion criteria. Subjects

requiring non-approved or excluded medication will not be eligible for enrolment.

Test product, dose and mode of administration, batch number:

FLUTCORE vaccine composed of VLP1 and VLP2 formulated in the presence/absence of

alhydrogel for intramuscular injection. The final volume of injection will not exceed 0.5mls.

The level of aluminium per dose will not exceed 1.25mg.

Duration of treatment:

The duration of the main study for each subject will be 12 weeks (84±2 days), comprising

vaccination on Days 0, 28 and 56, followed by a 4 week follow up period for analysis of

immunogenicity and safety. In addition, a screening visit will be conducted up to 4 weeks before

Day 0, and a follow-up telephone call will be conducted 6-months after the first dose for the

assessment of ongoing safety.

Reference therapy dose and mode of administration, batch number:

Placebo treatment will consist of Alhydrogel suspended in the same buffer as the IMP, administered

in a manner allowing full treatment blinding to both study personnel and subjects.

Criteria for evaluation (Study endpoints):

Safety and tolerability: Safety and tolerability will be assessed by comparing clinical laboratory

data pre- and post-vaccination and by the evaluation of adverse event profiles collected via the use

of diary cards rating specific signs/symptoms (solicited events) and by general questioning

(unsolicited events), plus physical examination.

Immunogenicity: Immunological responses will be measured at various time-points following

vaccination and compared between dose levels and groups, as detailed in the Study Event Schedule

below.

Statistical methods:

Adverse events will be summarized by dose, MedDRA (Medical Dictionary for Regulatory

Activities) coding, severity, and relationship to treatment. The descriptive statistics presented for

each system-organ class and preferred term will be the number of subjects with event (N), the

percent of subjects exposed with event (%), and the number of events (E). All adverse events will

be listed by subject no., dose, MedDRA system organ class, and MedDRA preferred term.

Clinical laboratory values, vital signs, and EKG will be listed. All values outside normal range (at

screening and at any follow-up visits) will be listed by subject no. and flagging of values.

Immunologic responses will be analysed by comparing the number of responders per group, as well

as the magnitude of the responses in the various groups. Fisher’s exact test, T-tests or Wilcoxon

tests will be used as appropriate.

Date: Written by

40

Table 10: Study event schedule

Study Event Schedule

Visit 1

Visit 2

Visit 3

Visit 4

Visit 5

Visit 6

Visit 7

Visit 8

Visit 9

Visit 10

Visit 11

Assessment D -28 to -1 Day 0 Day 1 Day 7 Day 28 Day 29 Day 35 Day 56 Day 57 Day 84 Day 183

Informed Consent X

Inc/Exc Criteria X

Demography X

Medical History X

Physical Examination X X X

Height, Weight, BMI X

Vital signs X X X X X X X X X X

12-lead ECG X X

Urinalysis X X

Safety laboratory tests X X X X X X X

HIV, Hep B and Hep C X

Pregnancy test X (serum) X (urine) X (urine) X (urine) X (urine)

Review of eligibility X X X

Randomisation X

Vaccination X X X

PD blood sample:

IgG/CMI

X X X X X X

Assess injection site X X X X X X X

AE check X X X X X X X X X X

Con med check X X X X X X X X X X X

41

Multidisciplinary question

Question 11

Does the agency have any other points (quality, non-clinical, clinical, regulatory) that the

company should consider in the development of this novel vaccine

42

6. List of references

1. CPMP Note for Guidance on Preclinical Pharmacological and Toxicological

Testing of Vaccines (CPMP/SWP/465/95)

2. Guideline on Clinical Evaluation of New Vaccines (CHMP/VWP/164653/2005)

3. Guideline on Influenza Vaccines - Non-clinical and clinical module

(EMA/CHMP/VWP/457259/2014)

4. WHO Guidelines on the nonclinical evaluation of vaccine adjuvants and adjuvanted

vaccines 2013.

5. Sweet C, Bird RA, Cavanagh D, Toms GL, Collie MH, Smith H. The local origin

of the febrile response induced in ferrets during respiratory infection with a virulent

influenza virus. Br J Exp Pathol. 1979 Jun; 60(3):300-8.

6. Belser JA, Katz JM, Tumpey TM. The ferret as a model organism to study

influenza A virus infection. Dis Model Mech. 2011 Sep; 4(5):575-9.

7. Zitzow LA, Rowe T, Morken T, Shieh WJ, Zaki S, Katz JM. Pathogenesis of avian

influenza A (H5N1) viruses in ferrets. J Virol. 2002 May; 76(9):4420-9.

8. Jackson S1, Van Hoeven N, Chen LM, Maines TR, Cox NJ, Katz JM, Donis RO.

Reassortment between avian H5N1 and human H3N2 influenza viruses in ferrets: a

public health risk assessment. J Virol. 2009 Aug; 83(16):8131-40.

9. Pearce MB, Jayaraman A, Pappas C, Belser JA, Zeng H, Gustin KM, Maines TR,

Sun X, Raman R, Cox NJ, Sasisekharan R, Katz JM, Tumpey TM. Pathogenesis

and transmission of swine origin A(H3N2)v influenza viruses in ferrets. Proc Natl

Acad Sci U S A. 2012 Mar 6; 109(10):3944-9.

10. Margine I, Krammer F. Animal models for influenza viruses: implications for

universal vaccine development. Pathogens. 2014 Oct 21; 3(4):845-74.