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SUPPLEMENTARY MATERIALS & METHODS
Methods Relating to VAC037 – Pilot Phase IIa Safety Study:
Study Design
The study was conducted at the Oxford Vaccine Centre, part of the Centre for Clinical
Vaccinology and Tropical Medicine, University of Oxford, UK with the challenge
procedure performed as previously described
1
using five infectious bites from
Plasmodium falciparum 3D7-strain infected Anopheles stephensi mosquitoes at the
Alexander Fleming Building, Imperial College, London, UK. All volunteers gave written
informed consent prior to participation, and the study was conducted according to the
principles of the Declaration of Helsinki and in accordance with Good Clinical Practice
(GCP). Approvals were granted by the UK Gene Therapy Advisory Committee (GTAC
166) and the UK Medicines and Healthcare products Regulatory Agency (Ref:
21584/0253/001-0001). The six control volunteers in the Phase IIa part of the VAC037
trial were recruited and consented as part of another study, MAL034B Group 7, with the
appropriate approvals described elsewhere (Ewer et al., submitted). Vaccine use was
authorized by the Genetically Modified Organisms Safety Committee (GMSC) of the
Oxford Radcliffe Hospitals NHS Trust (Reference number GM 462.09.43). The trial was
registered with ClinicalTrials.gov (Ref: NCT01003314). A Local Safety Monitor
provided safety oversight, whilst GCP compliance was externally monitored. The Phase
Ia part of this study has been reported elsewhere 2.
Three volunteers were vaccinated with 5 x 1010 vp ChAd63 MSP1 intramuscularly (IM)
(VAC037, Group 2C, see ref 2) and were subsequently vaccinated IM with 5 x 108 pfu
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MVA MSP1 56 days later. The three volunteers in group 2C, along with six unimmunized
control volunteers from the MAL034B study, underwent sporozoite CHMI 13-16 days
after the MVA MSP1 boost immunization. Volunteers attended for clinical review at days
2, 14, 28, 56, 58, 63 post ChAd63 MSP1 and the day before CHMI (dC-1). A time
window ranging between 1 and 14 days was allowed for vaccination and post vaccination
follow-up visits. Safety assessments, including blood sampling for vaccine safety and
immunology analysis at these visits were conducted as previously described 2. Post CHMI
volunteers were reviewed on dC+6.5 in the evening and then twice a day, morning and
evening between dC+7 and dC+14. Undiagnosed volunteers were reviewed once a day in
the morning between dC+15 and C+21. At each visit, blood was sampled for microscopy,
physical observations performed and AEs solicited. On diagnosis, volunteers were treated
with a 3 day curative course of oral Riamet where each dose was directly observed in
clinic. Volunteers intolerant of Riamet were prescribed an appropriate alternative (oral
Malarone or Cholorquine). Volunteers were reviewed 24 and 48 hours post diagnosis
where blood was sampled for microscopy. Provided these two blood-films were negative
for parasites volunteers were not reviewed again in clinic until dC+35. If one of these
blood films were positive, volunteers continued to be reviewed in clinic at 24 hour
intervals until two consecutive blood films were negative. Volunteers were then reviewed
at dC+35 and dC+90 where safety assessments, including blood sampling for safety and
immunology analysis at these visits were conducted.
Safety Assessment
From day 6.5 post challenge until day of diagnosis with malaria, volunteers in Group 2C
had daily measurement of full blood count with differential and platelet count. Serum
biochemistry (including electrolytes, urea, creatinine, bilirubin, alanine aminotransferase,
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alkaline phosphatase and albumin) was measured in volunteers in Group 2C on days 7
and 14 post-challenge or day of diagnosis if this was before day 14 post-challenge, and at
visits on dC+35 and dC+90.
Parasite Growth Modelling (Figs. S9a,b)
The number of infected erythrocytes in the first generation after parasite release from the
liver (liver-to-blood inoculum, LBI) and the parasite multiplication rate in the blood
(PMR) were estimated using qPCR data by application of a previously published
mathematical model fitted using Stata release 11 (Statacorp, Texas, USA). The model
based upon the normal cumulative density function (CDF) was fitted as previously
described by Hermsen et al. 3, with the following modifications: parameters µ1,2,3 and
σ1,2,3 were fixed using previously estimated values 3 – the same approach used in another
recent trial 4_ENREF_6; the PMR was estimated for each individual, rather than groups.
The model was not fitted to the single control subject (C4) for whom only two positive
qPCR data points were available. The linear and sine-wave models were fitted to the
VAC037 data as described below.
Methods Relating to VAC039:
Objectives
The objectives of this study (VAC039) were to assess the reactogenicity, safety,
immunogenicity and efficacy in healthy malaria-naïve adults of the ChAd63 and MVA
vaccines encoding AMA1 administered alone or co-administered with those encoding
MSP1, and the same for the vectors encoding MSP1 administered alone or coadministered with those encoding ME-TRAP.
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ChAd63 and MVA Vaccines
The vaccines were manufactured under Good Manufacturing Practice conditions by the
Clinical Biomanufacturing Facility, University of Oxford (ChAd63 vaccines) and IDT
Biologika, Rosslau, Germany (MVA vaccines). Each vaccine lot underwent
comprehensive quality control analysis to ensure that the purity, identity and integrity of
the virus met pre-defined specifications.
Study Design
This was a Phase I/IIa open-label, non-randomized vaccine and CHMI trial. Allocation to
study groups (Fig. 2) occurred at screening based on volunteer preference as previously
described 2. All vaccinations were administered intramuscularly (IM) into the deltoid. Ten
volunteers were vaccinated with 5 x 1010 viral particles (vp) ChAd63 MSP1 (undiluted
and administered in 360µL) and subsequently vaccinated in the opposite arm 56 days later
with 2 x 108 plaque forming units (pfu) MVA MSP1 (undiluted and administered in
210µL) (Group 1). Another nine volunteers were vaccinated with 5 x 1010 vp ChAd63
AMA1 (undiluted and administered in 300µL) and subsequently vaccinated in the
opposite arm 56 days later with 1.25 x 108 pfu MVA AMA1 (undiluted and administered
in 50µL) (Group 2). Another nine volunteers (Group 3) were vaccinated with ChAd63
MSP1 (as per Group 1) along with ChAd63 AMA1 (as per Group 2) co-administered at
the same time and into the opposite arm, followed 56 days later by MVA MSP1 (as per
Group 1) along with MVA AMA1 (as per Group 2) co-administered at the same time and
into the opposite arm (but each antigen was administered into the same arm for the prime
and the boost). A final ten volunteers (Group 4) were vaccinated in the same manner to
Group 3, but with ChAd63 MSP1 (as per Group 1) along with ChAd63 ME-TRAP 5 x
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1010 vp (undiluted and administered in 370µL), followed 56 days later by MVA MSP1 (as
per Group 1) along with MVA ME-TRAP 2 x 108 pfu (undiluted and administered in
230µL). Volunteers attended for clinical review at days 2, 14, 28, 56, 58, and 63 post
ChAd63 vaccines and the day before CHMI (dC-1). A time window ranging between 1
and 14 days was allowed for vaccination and post vaccination follow-up visits. Safety
assessments, including blood sampling for vaccine safety and immunology analysis at
these visits were conducted as previously described 2.
All volunteers in Groups 1-4 (bar one volunteer in Group 1), along with six unimmunized,
malaria naive control volunteers (Group 5 – different volunteers to the control volunteers
in VAC037) underwent sporozoite CHMI 14-18 days after the MVA boost immunization
(except two volunteers who were challenged 24 (Group 3) and 25 (Group 4) days after
MVA immunization). Post CHMI volunteers were reviewed on dC+6.5 in the evening and
then twice a day, morning and evening between dC+7 and dC+14. Undiagnosed
volunteers were reviewed once a day in the morning between dC+15 and C+21. At each
visit, blood was sampled for microscopy, physical observations performed and AEs
solicited. On diagnosis, volunteers were treated with a 3 day curative course of oral
Riamet where each dose was directly observed in clinic. Volunteers intolerant of Riamet
were prescribed an appropriate alternative (oral Malarone or Cholorquine). Volunteers
were reviewed 24 and 48 hours post diagnosis where blood was sampled for microscopy.
Provided these two blood-films were negative for parasites volunteers were not reviewed
again in clinic until dC+35. If one of these blood films were positive, volunteers
continued to be reviewed in clinic at 24 hour intervals until two consecutive blood films
were negative. Volunteers were then reviewed at dC+35, dC+90 and dC+150 where
safety assessments, including blood sampling for safety and immunology analysis were
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conducted. Throughout the paper, study day refers to the nominal time point for a group
and not the actual day of sampling.
Inclusion Criteria
•
Healthy adults aged 18 to 50 years.
•
Able and willing (in the Investigator’s opinion) to comply with all study
requirements.
•
Willing to allow the investigators to discuss the volunteer’s medical history with
their General Practitioner.
•
For female volunteers, willingness to practice continuous effective contraception
for the duration of the study.
•
Agreement to refrain from blood donation during the course of the study.
•
Written informed consent.
Exclusion Criteria
•
History of clinical P. falciparum malaria.
•
Travel to a malaria endemic region during the study period or within the preceding
six months with a risk of malaria exposure.
•
Participation in another research study involving an investigational product in the
30 days preceding enrolment, or planned use during the study period.
•
Prior receipt of an investigational malaria vaccine or any other investigational
vaccine likely to impact on interpretation of the trial data.
•
Administration of immunoglobulins and/or any blood products within the three
months preceding the planned administration of the vaccine candidate.
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•
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Any confirmed or suspected immunosuppressive or immunodeficient state,
including HIV infection; asplenia; recurrent, severe infections and chronic
(more than 14 days) immunosuppressant medication within the past 6 months.
•
Pregnancy, lactation or intention to become pregnant during the study.
•
Contraindication to both anti-malarial drugs; Riamet & chloroquine.
•
Concomitant use of other drugs known to cause QT-interval prolongation (e.g.
macrolides, quinolones, amiodarone etc).
•
History of epilepsy.
•
History of arrhythmia or prolonged QT interval.
•
Family history for sudden cardiac death.
•
An estimated, ten year risk of fatal cardiovascular disease of ≥5%, as estimated by
the Systematic Coronary Risk Evaluation (SCORE) system 5.
•
History of allergic disease or reactions likely to be exacerbated by any component
of the vaccine e.g. egg products, Kathon.
•
History of clinically significant contact dermatitis.
•
Any history of anaphylaxis post vaccination.
•
History of cancer (except basal cell carcinoma of the skin and cervical carcinoma
in situ).
•
History of serious psychiatric condition that may affect participation in the study.
•
Any other serious chronic illness requiring hospital specialist supervision.
•
Suspected or known current alcohol abuse as defined by an alcohol intake of
greater than 42 units every week.
•
Suspected or known injecting drug abuse in the 5 years preceding enrolment.
•
Seropositive for hepatitis B surface antigen (HBsAg).
•
Seropositive for hepatitis C virus (antibodies to HCV).
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Any clinically significant abnormal finding on biochemistry or haematology blood
tests or urinalysis.
•
Any other significant disease, disorder or finding which may significantly increase
the risk to the volunteer because of participation in the study, affect the ability
of the volunteer to participate in the study or impair interpretation of the study
data.
Safety Assessment
The first volunteer to receive each dose of the co-administered ChAd63 or MVA vaccines
(in Groups 3 and 4) was vaccinated in isolation. Following a review of reactogenicity in
these individuals 48 hours post the priming and boosting vaccinations, the remaining
volunteers in these two groups were vaccinated.
Volunteers in Groups 1 and 2 were observed for 1 hour post ChAd63 vaccines and 30
minutes post MVA vaccines. Volunteers in Groups 3 and 4 were observed for 1 hour post
each immunization. Volunteers were given a digital thermometer, injection site reaction
measurement tool and symptom diary card to record their daily temperature, injection site
reactions and solicited systemic AEs for 14 days following vaccination with ChAd63
vectored vaccines and 7 days following vaccination with MVA vectored vaccines. Local
and systemic reactogenicity was evaluated at subsequent clinic visits and graded for
severity, outcome and association to vaccination as per the criteria outlined in Tables S46. Blood was sampled at all visits post vaccination except days 2 and 58. Full blood count
with differential, platelet count and serum biochemistry (including electrolytes, urea,
creatinine, bilirubin, alanine aminotransferase, alkaline phosphatase and albumin) were
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measured at all visits before CHMI (except days 2 and 58), at visit dC+9, within 24 hours
of diagnosis, and at visits on dC+35, dC+90 and dC+150.
Peptides for T cell Assays
Peptides were used to assess T cell responses as previously described for each antigen
2,6,7
, with some minor modifications as follows: For MSP1 and AMA1, a number of minor
changes were made to the peptides previously used to ensure peptide sequences better
corresponded to the blocks of sequence encoded within the composite vaccine transgene
inserts. Similarly, a number of new peptides were designed to assess responses to de novo
sequence created at the junctions where sequences from different regions of the MSP1 or
AMA1 molecules were joined together. New peptides were purchased from Peptide
Protein Research Ltd (Funtley, Fareham, United Kingdom) and these as well as the
changes are detailed in Table S1. Peptides were reconstituted in 100% DMSO at 50-200
mg/mL and combined into various pools for ELISPOT and flow cytometry assays. For exvivo interferon-γ (IFN-γ) ELISPOT assays the peptides were divided into pools
(containing up to 10 peptides per pool, with the exception of the ME pool which contains
20 peptides) and the data show the summed total response for the MSP1 vaccine insert as
previously described 2, the 3D7 allele-specific response for AMA1 (3D7-specific peptides
+ common peptides + C-terminal tail peptides) 7, and for ME-TRAP the summed response
to vaccine heterologous / challenge homologous 3D7 sequence TRAP peptides plus those
encoding the ME string 6. For flow cytometry assays, the peptides were pooled into single
pools containing 108 peptides spanning the entire MSP1 vaccine antigen, a pool of all 56
peptides spanning the 3D7 allele AMA1 vaccine antigen 7, and a pool of all 57 peptides
encoding the 3D7 allele TRAP antigen sequence 6.
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Parasite Growth Modelling (Figs. 7, S7, S8a,b)
The number of infected erythrocytes in the first generation after parasite release from the
liver (liver-to-blood inoculum, LBI) and the parasite multiplication rate in the blood
(PMR) were estimated using qPCR data by application of two mathematical models. Both
models were fitted using Stata release 11 (Statacorp, Texas, USA). All qPCR values <20
parasites / mL were treated as negative. Negative values prior to any positive value were
disregarded (treated as missing) for linear and sine wave modelling; negative values
preceded by positive values were retained for modelling given they constitute useful
information – these were assigned an arbitrary value of 0.1. Prior to the fitting of both
models, 10 was added to all qPCR values (i.e. half the detection limit of the qPCR method
was added to the values to avoid giving too much weight to values near zero 3) and all
were transformed by taking log10. A 0.3 day (i.e. 7.2 hour) interval was assumed between
morning and afternoon/evening blood-sampling times.
Model 1 (Fig. 7): A sine-wave based model was fitted to individuals as described by
Bejon et al. 8:
Sine wave: log10(pcr+10) = c + m(day-6.5) + a[sin [π*(day-6.5) + k]
Model 2 (Fig. S7): A linear model was fitted to individual volunteers’ qPCR data:
Straight line: log10(pcr+10) = m(day) + c
Of the total 9 volunteers in VAC037, all had ≥5 positive qPCR data points (Table S2)
with the exception of one control subject (C4), who had only two positive qPCR data
points prior to diagnosis. The sine wave model was not fitted for this subject. Of total 42
volunteers in VAC039, all had ≥5 positive qPCR data points (Table S3) with the
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exception of the single sterilely protected volunteer (G3-9). Thus both models could be
fitted to all volunteers.
LBI was estimated in four ways:
A.
Maximum day 7 or day 7.5 qPCR value for each volunteer.
B.
Total qPCR-measured LBI (output A corrected for estimated blood volume using
70mL/kg body mass). Clinical Anaesthesiology by Morgan Mikhail and Murray
estimates 75mL/kg for men and 65mL/kg for women 9. For VAC037 volunteers, a
body mass of 70kg was assumed; for VAC039 volunteers, body mass was as
measured. Negative qPCR results on day 7 and day 7.5 for volunteers who
developed blood-stage infection are graphically represented as 10,000 total liver
emerging parasites (Fig. S7a).
C.
LBI estimated from linear model: output when day = 7.5.
D.
LBI estimated from sine wave model = 10|a|+c 8.
PMR was estimated in two ways:
E.
PMR, derived from linear model-fitted parameter ‘m’: 102m
F.
PMR derived from sine wave model-fitted parameter ‘m’: 102m
The pre-specified per protocol primary modelling outcome measures for VAC039 was the
sine-wave model (outputs D and F, reported in Fig. 7). We also report weight-corrected
total LBI (output B) and the PMR both calculated from the linear model (output E). These
outputs are reported for both trials in Fig. S7. The correlation between these two outputs
for LBI and PMR is reported in Fig. S8a,b. A more detailed comparison of these
modelling methods is described elsewhere (Douglas AD et al., manuscript in preparation).
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