View
8
Download
0
Category
Preview:
Citation preview
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.
Recommended