gmz2 phase2b statistical analysis plan v1p a g e | 1 of 19 gmz2 phase iib statistical analysis plan...

P a g e | 1 of 19 GMZ2 Phase IIb Statistical Analysis Plan V1.2 Mar 16th 2015

A phase IIb, randomized, controlled, double-blind, multi-centre study to evaluate the efficacy, safety, and immunogenicity of GMZ2 candidate malaria vaccine in Gabonese, Burkinabe, Ghanaian and Ugandan children aged 12 – 60 months.

Statistical Analysis Plan

Version 1.1 Oct 6 2012

Version 1.2 Mar 16th 2015

Paul Milligan Faculty of Epidemiology and Population Health London School of Hygiene and Tropical Medicine October 6th 2012


Protocol number Protocol 1.0 amendment 1.1

Protocol version Protocol version 1.1

Study title A phase IIb, randomized, controlled, double-blind, multi-centre study to

evaluate the efficacy, safety, and immunogenicity of GMZ2 candidate

malaria vaccine in Gabonese, Burkinabe, Ghanaian and Ugandan children

aged 12 – 60 months.

Phase IIb

Vaccine Active arm: Three doses of 100µg lyophilized recombinant Lactococcus

lactis hybrid GMZ2 [GLURP+MSP3], adjuvanted in aluminium hydroxide,

28 days apart, administered Intramuscular. Control arm: three doses of

human diploid cell (HDC) rabies vaccine administered intramuscularly.

Authors V1.1 Paul Milligan

V1.2 Paul Milligan and Clare Flach

List of investigators PIs: Sodiomon Sirima, Saadou Issifou, Fred Kironde, Frank Atuguba

Co-PIs: Alfred Tiono, Ateba Ulysse Ngoa,Kalifa Bojang, Mark

Kaddumukasa, Abraham Hodgson Collaborators: Michael Thiesen,

Benjamin Mordmüller, Roma Chilengi, Søren Jepsen

Data Management: Abubakar Ismaela

Sponsor: Staten Serum Institut

Trial coordination for sponsor: Dawit A. Ejigu, Brenda Okech

GCP compliance The study is conducted according to current International Conference of

Harmonization Good Clinical Practices (ICH-GCPs) and the applicable

regulations of Ghana, Uganda, Burkina Faso and Gabon.

Version number 1.2

Date of this document 16 Mar 2015

Signatures Independent Biostatistician

Principal Investigators and PSC members

Sponsor

DSMB Chairperson


Contents:

1. Study sites

2. Study population

3. Primary objective

4. Secondary objectives

5. Exploratory objectives

6. Primary endpoint

7. Secondary endpoints

8. Database

9. Unblinding

10. Trial profile

11. Loss to follow-up

12. Missing data

13. Population at risk

14. Protocol deviations

15. Baseline characteristics

16. Vaccine administration

17. Analysis populations

18. Safety and reactogenicity

19. Biochemistry and haematology parameters

20. Immunogenicity

21. Efficacy

22. Surrogates of protection

23. Dissemination plan

24. Technical notes

25. References

26. Individual patient listings

27. Reference ranges and AE gradings


Study sites: Iganga, Uganda; Lambaréné, Gabon; Banfora, Burkina Faso; Sapone, Burkina Faso; Navrongo,

Ghana.

Study population: healthy children, boys and girls, aged 12-60 months at first vaccination.

Primary objective:

To assess the efficacy of GMZ2 candidate vaccine against Plasmodium falciparum clinical malaria episodes over

a six months surveillance period starting from the day of the third dose of vaccination. To permit ITT analysis of

efficacy, surveillance for malaria will start immediately after randomization

Secondary objectives:

To evaluate the safety and reactogenicity of three doses of GMZ2 candidate malaria vaccine adsorbed on

aluminium hydroxide.

To assess the efficacy of GMZ2 in preventing anaemia (Hb < 7g/dl) and severe anaemia (Hb < 5g/dl) assessed

at 6 months after vaccination (D 252).

To assess efficacy of GMZ2 against fever (defined as fever and/or history of fever) with parasitemia (using 4

threshold densities, at any density above zero, and at least 500, 2500 and 20,000 trophozoites/µL) over 6

months and at densities of any density above zero, and at least 500, 2500, 5000 and 20,000/µL, over 12 and 22

months.

Exploratory objectives:

To evaluate humoral responses to the vaccine antigens by measuring the level of antigen specific IgG on Days

0, 28, 56, 84, 168, 252, 365, 588 and 730, and, in a subset of participants, antigen specific memory B-cell by

ELISPOT on Days 0, 84, 252, 365 and 730.

To evaluate cellular immune responses by measuring T-cell IFN-γ production on single cell level and Th1/Th2

cytokine profiling after stimulation with the vaccine antigens.

To assess the functionality of the immune response by measuring the Growth Inhibition of P. falciparum in the

presence or absence of Monocytes.

To assess cell-mediated immunogenicity by cytokine profiling and intracellular cell staining following

stimulation with the vaccine antigen.

To assess the quality of the antibody response by type and subclass specific ELISA and posttranslational

antibody modifications.

To estimate the effect of other infections on immune response and vaccine efficacy.

To explore biomarkers as surrogates of vaccine efficacy.

To assess changes in parasite populations induced by the GMZ2 vaccine by comparing parasite populations

before vaccination as well as in the two arms by assessing the genotype by PCR and DNA sequencing of MSP3

and GLURP genes and growth inhibition of cryopreserved clinical isolates by GIA.

To investigate evidence for waning of efficacy.

To evaluate the potential effect of GMZ2 on Gametocyte carriage by microscopic evaluation of blood smear on

days 0, 84, 365 and 588.

To estimate efficacy against severe malaria, all-cause hospital admission and all cause mortality.


Primary evaluation criteria:

A clinical case of malaria will be defined as a participant who is unwell presenting with fever ≥ 38°C measured

by the tympanic method, or a history of fever in the previous 48 hours, with parasite density ≥ 5000

trophozoites/µL. Efficacy will be assessed over a period starting at dose 3 and ending 6 months after dose 3

(ATP), and starting at randomization and ending 6 months after scheduled time for dose 3 (ITT), provided at

least 330 children have had an episode of malaria that meets the primary case definition during the ATP

period. If this criterion is not met, the analysis time period will be extended until 330 first episodes in the ATP

follow up period have accrued, up to a maximum of 12 months post dose 3. If 330 first episodes have not

accrued by 12 months, the analysis will be done at 12 months. The total period of follow-up will be 24 months

from the first dose of vaccine.

Secondary evaluation criteria:

Immediate reactogenicity (reactions within 30 minutes after each injection, with emphasis on allergic reaction)

Local and systemic reactogenicity measured from Day 0 to Day 7 after each dose

Any adverse event resulting in a visit to a physician between each injection and one month after the third dose

Any Serious Adverse Event (SAE) occurring from signing of the informed consent and throughout the trial.

Anaemia (Hb < 7g/dl) and severe anaemia (Hb < 5g/dl) at D252.

Severe malaria as defined in the Clinical Trial Protocol.

Hospital admission: admission for any cause for at least 24hours.

Hospital admission with malaria as the primary diagnosis.

Deaths from all causes.


Database: In each site, when the database is ready (double entry completed up to 6 months post dose 3),

laboratory data are complete, and queries have been resolved, a series of consistency checks will be run to

check: missing data, duplications, ID errors, linkage between tables, values out of range, and consistency of

dates. Any queries will be resolved and then a monitoring visit completed, before the database is finalised and

locked. Data in OpenClinica will be extracted into Access files for analysis using the same standard format for

all sites. A copy of the Access database will then be archived and then the code will be linked to the database

by the trial statistician who will implement the analysis plan.

As advised following a trial audit, data from visit 2 onwards were re-entered. All CRFs were sent to Tübingen

University (UKT) where they were entered into a database set up by Larix A/S in eClinical eDM and eDC system

version 5.0. Screening booklets (Visit 1) were not collected. Single data entry was used. The verification of the

data entry consisted of proof reading of selected areas combined with full proof reading of single CRFs to

verify the quality of the data entry in general. See also the data management file for the study for details. The

validation of data was done by Larix and UKT according to the approved validation plan and consisted of

programmed consistency checks in the database (Larix), programmed checks programmed in SAS (Larix), and

manual review of the paper CRFs (for missing pages and in-consistencies in page headers) (UKT). The trial

statisticians were not involved in the data cleaning and will be sent a final data set in SAS software, copied to

the DSMB on 17th March 2015.

Data analysis will be carried out using Stata version 13.1.

Unblinding: The investigators will remain blind to vaccine allocation until the end of the trial. To preserve

blinding, the statistician will ensure that the data that are potentially unblinding (individual data, immunology

results linked to study ID, and event outcomes that occurred in one vaccine group only) will be excluded from

the statistical report. Investigators (except those involved in laboratory analysis) will see immunology results

only in summary form.

Trial profile: Number screened, number eligible, reasons for non-eligibility, number enrolled, reasons for non-

enrolment, number that received dose 1,2,3, number that were withdrawn, died or were lost to follow up, will

be given with reasons for loss to follow-up or withdrawal.

Loss to follow-up: rates of known drop out will be compared between trial groups and with respect to baseline

characteristics. Rates of non-participation in cross-sectional surveys (i.e. visits that take place at the same

fixed times for all participants) will be similarly investigated.

Missing data: the proportion of observations with missing values will be summarised for important variables.

Missing periods of follow-up, determined by absence from cross-sectional surveys and clinic visits, will be

tabulated by site and vaccine group.

Population at risk: for analysis of malaria incidence, participants will be assumed to be at risk unless they are

known to have died or left the study area; observations will be censored at the time of death or emigration.

Protocol deviations: Eligibility errors; prior treatments and vaccinations; concomitant treatments and

vaccinations; randomization errors; vaccine administration errors; dose intervals outside range; Anti HB,

immunological assays, blood samples not taken; Alkaline phosphates not measured in Burkina Faso and

Gabon.

Table: of concomitant medications:

Date Days since most

recent dose

Subject ID Medication

or vaccine

Dose and

duration

Vaccine

group

Site

X days after dose n

…


Medical History at screening

SYSTEM / ORGANS STATUS

Current Past

Eyes

Ear, Nose, Throat

Cardiovascular

Respiratory, Thoracic and Mediastinal

Gastrointestinal

Renal and Urinary

Nervous system

Immune System

Hepatobiliary

Musculoskeletal

Infections/ infestations

Neoplasms benign, malignant & unspecified

Other (specify)

Enrollment

Participants GMZ2 Rabies Overall

Planned enrollment

Screened and randomized

Included in the safety analysis

Included in the primary efficacy analysis

Included in the secondary efficacy analysis

Included in the immunological analysis

Baseline characteristics:

Table of baseline characteristics from visit 1 (by randomized group), all sites combined:

Variable GMZ2 Control

Number randomized

Age in months (mean,range)

Age group (number (%) by year of age)

Gender

Bednet use (use of any net, and use of ITN)

Temperature (mean, range)

Weight in kg (mean, range)

Weight for age z-score (mean, %<-2) (WHO 2006 standards)

Height for age (mean, %<-2)

Weight for height (mean, %<-2)


− Hb concentration g/dL (mean, range)

Biochemical and haematological variables (mean, range, % outside normal range):

Creatinine

ALT

Bilirubin

White Blood Cell

Red blood Cell

Platelets

ASAT

Alkaline Phosphate

Haemoglobin

Haematocrit

Immunological variables:

Total IgG, and IgG subclasses, to GMZ2, to GLURP, and to MSP3

Sickle cell genotype HbS

G6PD genotype

P. falciparum % positive for asexual parasites

% positive for gametocytes

Arithmetic and geometric mean density of asexual and gametocytes (range)

Dates of vaccination (first and last to be vaccinated in each site) for dose 1 and dose 3

In addition, a similar table of baseline characteristics will be produce for each site.

Vaccine administration: Dose timings, Interval between doses, dose errors.

Dose timings: date of first and last child vaccinated

Banfora Sapone Iganga Lambarene Navrongo

Dose 1

Dose 2

Dose 3

Doses received:

GMZ2 Control

Doses received:

1

2

3

1,2

1,3

2,3

1,2,3

Dose intervals:

Median (min, max) interval (days)


Dose 1 to Dose 2

Dose 2 to Dose 3

Vaccination errors: individual listing.

Reasons for discontinuation

GMZ2 vaccine Rabies vaccine Overall

n (%) n (%) n (%)

− Number of subjects who

discontinued

Reasons for discontinuation

Non serious adverse event

Serious adverse event

Protocol violation

Consent withdrawal

Migration from the study area

Lost to follow up (after complete vaccination)

Lost to follow up (before complete vaccination)

Other

Analysis populations:

Intention to treat (ITT) for efficacy: ITT analyses will be done including all children who were randomized, in

the group they were randomized to.

According to Protocol (ATP) for efficacy: All children who received three vaccine doses. Children who had

concomitant vaccination or medication prohibited by the protocol will be included in the primary ATP analysis.

A secondary ATP analysis will be done excluding these individuals and any individuals enrolled who did not

meet eligibility criteria, and exploring the effect of out-of-range dose intervals. Children who received the

wrong vaccine in error will be included according to the vaccine they received, if all doses were of the same

vaccine type, if they received a mixture they will be excluded from the ATP analysis.

For safety endpoints, all children who received vaccine doses will be included according to the vaccine they

actually received.

For immunogenicity, analysis will include all children whose responses were measured after all of the three

doses. A secondary analysis will investigate effects of having received concomitant medications or

vaccinations.

For surrogates of protection, all children in the ATP population for efficacy will be included provided they had

immunology measurements post dose 3.

[The ITT analysis aims to keep as closely as possible to the randomization, to obtain the fairest estimate of

efficacy, so all subjects are included and the surveillance period starts from the time of randomization. It is

anticipated that three doses are needed for maximum protection so including follow-up time before dose 3

could under-estimate the protective effect, but there could be a benefit of one or two doses which would be

missed in the ATP analysis, furthermore in the unlikely event that the vaccine caused malaria episodes, and

this caused individuals to drop out, this would be missed if surveillance started later. ATP population: the main


purpose is to limit analysis to children who received the full course of vaccinations in order to better estimate

the magnitude of the effect in fully vaccinated children, it is undesirable to exclude children with protocol

violations unless it is known these are likely to reduce efficacy, therefore all children with 3 doses will be

included and a secondary analysis will be done to explore the effect of excluding children with protocol

violations (concomitant vaccination or drug therapy).]


Safety and reactogenicity:

Individuals who received mixed doses in error (e.g. GMZ2 at dose 1 and rabies vaccine at dose 2 etc) will be

analysed separately. All other individuals will be included according to the vaccine they received.

Serious adverse events will be listed individually, with investigator assessment of relationship to vaccination.

For any event and then separately for each type of event, the relative risk (GMZ2:control) (for any severity,

and for grade 3 only) of an event during the period from dose 1 up to 28 days after dose 3 will be calculated

with a 95% confidence interval. Adverse events will be sorted by relative risk and the relative risk of each

solicited adverse event displayed in a forest plot with confidence intervals. For common adverse events,

frequency of any event, and of a grade 3 event, during the vaccination period will be tabulated by age group.

Serious adverse events:

Date Site Age,

gender

Vaccine

group

Time

since

most

recent

dose

Description Action

taken

Outcome Investigator

assessment of

relationship to

vaccination*

(Relationship to vaccination: 1) most probably related, 2) probably related, 3) possibly related, 4) unlikely to be

related, 5) not related, 6) insufficient evidence to assess relationship).

Adverse events during the 7 days follow up period, by dose and overall for all the participants

GMZ2 Rabies

Dose 1

Any adverse event

Local

General

Dose 2

Any adverse event

Local

General

Dose 3

Any adverse event

Local

General

Any dose

Any adverse event

Local

General


Time of occurrence of local solicited adverse events after vaccination by dose, vaccine group

GMZ2 Rabies

Dose 1 Dose 2 Dose 3 Any dose Dose 1 Dose 2 Dose 3 Any dose

Pain at injection site

Any time

Immediate period post

immunization

Day 0

Day 1

Day 2

Day 3

> day 3

Swelling

Any time


immunization

Day 0

Day 1

Day 2

Day 3

> day 3

Induration

Any time


immunization

Day 0

Day 1

Day 2

Day 3

> day 3

Erythema

Any time


immunization

Day 0

Day 1

Day 2

Day 3

> day 3

Induration

Any time


immunization

Day 0

Day 1

Day 2

Day 3

> day 3

Pruritus


Any time


immunization

Day 0

Day 1

Day 2

Day 3

> day 3

Time of occurrence of systemic solicited adverse events after vaccination by dose, vaccine group

GMZ2 Rabies

Dose 1 Dose 2 Dose 3 Any dose Dose 1 Dose 2 Dose 3 Any dose

Fever

Any time


immunization

Day 0

Day 1

Day 2

Day 3

> day 3

Irritability

Any time


immunization

Day 0

Day 1

Day 2

Day 3

> day 3

Drowsiness

Any time


immunization

Day 0

Day 1

Day 2

Day 3

> day 3

Loss of appetite

Any time


immunization

Day 0

Day 1

Day 2

Day 3

> day 3


Loss of appetite

Any time


immunization

Day 0

Day 1

Day 2

Day 3

> day 3

Solicited local symptoms: (within 7 days of the dose)

GMZ2 Rabies vaccine

Dose1 Dose2 Dose3 After

any

dose


any

dose

Relative

Risk after

any dose

(95%CI)

Any

solicited

local

adverse

event

Number

affected

(%)

Any

Grade 3

Pain

Any

Grade 3

Swelling

Any

Grade 3

Induration

Any

Grade 3

Erythema

Any

Grade 3

Pruritus

Any

Grade 3


Solicited general symptoms: (within 7 days of the dose)

GMZ2 Rabies vaccine


any

dose


any

dose

Relative

Risk after

any dose

(95%CI)

Any

solicited

general

adverse

event

Number

affected

(%)

Any

related

Fever

Any

related

Irritability

Any

related

Drowsiness

Any

related

Loss of

appetite

Any

related

Solicited grade 3 general symptoms: (within 7 days of the dose)

GMZ2 Rabies vaccine


any

dose

Relative

Risk after

any dose

(95%CI)


any

dose

Relative

Risk after

any dose

(95%CI)

Any

solicited

general

adverse

event

Number

affected

(%)

Any grade 3

Grade 3

related

Fever

Any grade 3

Grade 3

related

Irritability

Any grade 3

Grade 3

related


Drowsiness

Any grade 3

Grade 3

related

Loss of

appetite

Any grade 3

Grade 3

related

Unsolicited adverse events by vaccine group and dose symptoms (within 28 days of the dose):

GMZ2 Rabies vaccine


any

dose


any

dose

Any unsolicited

adverse event

Number

affected

(%)

Any

Related

Body System

Preferred term

Unsolicited grade 3 adverse events by vaccine group and dose

GMZ2 Rabies vaccine


any

dose


any

dose

Any unsolicited

adverse event

Number

affected

(%)

Any

Grade 3 related

Body System

Preferred term

Grade 3 related


Biochemistry and haematology parameters:

Measurements of biochemical and haematological parameters after each dose of vaccine will be compared

between GMZ2 and control groups, using analysis of covariance, with the pre-dose value as a covariate.

Dose 1 Dose 2 Dose 3

Variable Time

point

GMZ2 Rabies vaccine GMZ2 Rabies vaccine GMZ2 Rabies vaccine

Creatinine Pre

Post

Δ

ALT Pre

Post

Δ

Hb Pre Post Δ

Haematocrit Pre Post Δ

Platelets Pre Post Δ

WBC Pre Post Δ

RBC Pre Post Δ

The number of children with values outside the normal range will be tabulated before and after each dose.


Immunogenicity:

Concentration of total IgG, and IgG subclasses, to GMZ2, GLURP and MSP3 will be analysed. Zero values for

antibody concentrations will be replaced with a constant value equal to half the smallest non-zero

concentration for that variable in the dataset. Antibody concentrations will be transformed to logarithms for

analysis. Data will be summarised by the arithmetic or geometric mean at each time point (with range). To

compare responses between the vaccine groups, geometric means in the two vaccine groups will be compared

at each time point using analysis of covariance (with baseline concentration as a covariate), with a 95% CI for

the geometric mean ratio between groups. Responses will be compared by age group (in integer years), and by

site. Plots of concentration over time showing individual responses will be produced.

[Analysis of change from baseline: rather than calculate the change from baseline, and then compare these

changes between groups, it is generally recommended to use a regression model to estimate the difference

between the groups, and adjust for the baseline by including the baseline measurement as a covariate. The

reason is that analysis of changes from baseline over-corrects for baseline imbalance (Senn, 1997). ]

Post data collection note: although this was the method of analysis originally planned, the assays were run at a

high dilution resulting in a high proportion of zero values. Geometric means are therefore problematic and

would require imputing a large number of values. Therefore, we will do two analyses, a) calculate arithmetic

means and compare these between groups using poisson regression with a robust standard error b) compare

the proportion positive.

Table format for antibody responses: A separate table will be produced for total IgG to GMZ2, GLURP and

MSP3, and for each IgG subclass. These tables will be produced using pooled data, and then separately for

each site.

a) comparing arithmetic mean

GMZ2 Rabies vaccine Ratio between vaccine

groups (95%CI),

adjusted for baseline

Arithmetic

mean (N)

95%CI

ratio to D0

value

Arithmetic

mean (N)

95%CI

ratio to D0

value

GMZ2:Rabies vaccine

Day 0 pre-vacc1 1 1

Day 28 (V1+28)

Day 56 (V2+28)

Day 84 (V3+28)

Day 168 (V3+4mth)

Day 252 (V3+7mth)

Day 365 (V3+11mth)

Day 588 (V3+19mth)

Day 730 (V3+24mth)

b) comparing proportion positive

GMZ2 Rabies

vaccine

Prevalence ratio between vaccine

groups (95%CI), adjusted for

baseline

Positive IgG

N (%)

Positive IgG

N (%)

GMZ2:Rabies vaccine

Day 0 pre-vacc1

Day 28 (V1+28)

Day 56 (V2+28)

Day 84 (V3+28)

Day 168 (V3+4mth)

Day 252 (V3+7mth)


Day 365 (V3+11mth)

Day 588 (V3+19mth)

Day 730 (V3+24mth)


Efficacy:

The overall measure of efficacy will be the percentage reduction in the total number of episodes across all

sites estimated using Cox regression.

Time periods: For the ATP analysis, the surveillance period starts on the day of dose 3 and ends 6 months later.

For the ITT analysis, the surveillance period starts at randomization. The ATP and ITT analyses will be

presented, stratified by site but otherwise unadjusted for covariates.

Analyses will be repeated under the ITT and ATP definitions but extending the surveillance period in both cases

to the end of follow-up at 24 months from the first vaccination.

Incidence rates: For each case definition, the incidence rate of malaria will be calculated as the number of

episodes meeting the case definition divided by the time at risk. Time at risk will be the total time from start to

end of the surveillance period with no deduction for periods of malaria treatment. To avoid counting the same

episode twice, cases occurring within 14 days of a previous case will be ignored. Returns to clinic with malaria

symptoms within 14 days of the primary case are considered to be a single episode of malaria. Returns after 14

days are considered a separate illness episode, whether this is due to recrudescence or re-infection is

unknown but is immaterial as it is an attack of malaria not prevented by the vaccine.

Cumulative incidence, i.e. the proportion of children who have at least one episode, with 95% confidence

interval, will be estimated by the Kaplan Meier method.

The incidence rate of malaria will be compared between groups using Cox regression, using time since

randomization (ITT), or time since dose 3 days (ATP), as the time scale, and including all malaria episodes.

Vaccine efficacy (VE) will be defined as 100x(1-HR), where HR is the hazard ratio from Cox regression, this is an

estimate of the percentage reduction in the number of malaria episodes due to vaccination (Cheung et al.

2010). A 95% confidence interval will be calculated, using a robust standard error to allow for repeated

episodes in the same child. The analysis will be pooled across sites, with site as a stratification factor to allow

for a separate seasonal pattern of incidence in the control group in each site.

Vaccine attributable reduction (VAR) will be calculated as the difference incidence rates between the two

vaccine groups. This is a measure of impact which provides an estimate of the number of malaria episodes

prevented, the VAR will be estimated by the method of Xu et al. (2010), adjusted for site, using a robust

standard error to allow for repeated episodes in the same child.

The number (%) of children with 0,1,2,3,... episodes will be tabulated by vaccine group.

Covariate adjustment: The primary analysis will be unadjusted for covariates. A secondary analysis will adjust

for age at randomization, and ITN use at baseline.

These analyses will be repeated for a range of parasite density cut-offs.

A further analysis will estimate efficacy separately in each site.

To investigate variation in efficacy with time since vaccination, the number of episodes and the time at risk will

be tabulated for each month of follow up. Variation in efficacy with time will be assessed using flexible

regressions splines (Royston and Lambert). The final analysis will include efficacy estimates throughout the

duration of trial follow-up.

Subgroup analyses for efficacy: subgroups of interest, which will be analysed using tests for interaction in the

Cox model for the ATP analysis adjusted for covariates, are: Site; ITN use recorded at baseline; age at baseline.

Effect of antimalarial treatments: To investigate the possible bias due to prophylactic effect of intake of drugs

with antimalarial effect during the surveillance period, a) the number of antimalarial treatments (antimalarial

drugs and antibiotics with antimalarial action) in the two groups, and b) vaccine efficacy will be estimated, in

an exploratory analysis, after including a time-dependent covariate for the effect of each drug treatment on


malaria incidence. The duration of effect will assumed to be, for Coartem (14) days, for cotrimoxazole (21

days), azithromycin (7 days).

Parasitaemia: the prevalence of parasitaemia will be compared at each time point, the prevalence ratio with

95% confidence interval will be presented for each site and in a pooled analysis, the prevalence ratio will be

estimated using a generalised linear model with identity link. Prevalence of anaemia on day 252 will be

similarly analysed. Parasite densities (asexual, and gametocytes) will be tabulated at each time point and

compared using arithmetic means, analysed using Poisson regression with robust standard error at each time

point and in a combined analysis using all time points.


Vaccine efficacy tables:

Fever/history of fever and parasite density >5000/uL: ITT

GMZ2 Rabies

Vaccine

VE

95%CI)

Rate (No episodes/PYAR):

Banfora

Sapone

Iganga

Lambarene

Navrongo

Pooled

Fever/history of fever and parasite density >5000/uL: ATP

GMZ2 Rabies

Vaccine

VE

95%CI)


Banfora

Sapone

Iganga

Lambarene

Navrongo

Pooled

Vaccine efficacy by month of follow-up: primary case definition (ITT)

Incidence rate (no episodes/PYAR)

Month post dose 1: GMZ2 Rabies vaccine VE (95%CI)

1

2

3

….

Vaccine efficacy by month of follow-up: primary case definition (ATP)


Month post dose 3: GMZ2 Rabies vaccine VE (95%CI)

1

2

3

….

Vaccine efficacy by cutoff (ATP):


GMZ2 Rabies vaccine VE (95%CI)

Fever and any parasitaemia

Fever and parasite density >500/uL






Fever or history of fever and any parasitaemia

Fever or history of fever and parasite density >500/uL





Hazard ratios adjusted for covariates (ATP):

Incidence rate Crude HR (95%CI)# CrudeHR(95%CI)## Adjusted HR(95%CI)###

Vaccine group: GMZ2 1 1

Rabies Vaccine

Age at enrolment: 12-23 mths 1 1

24-35 mths

36-48mths

49-60mths

ITN use: Yes 1 1

No

# Includes all children in the ATP population who have data for the covariate

## Unadjusted for covariates but the analysis limited to the subset of children who have non-missing data for all covariates

### Adjusted for covariates

Severe malaria: ITT

GMZ2 Rabies

Vaccine

VE

95%CI)


Banfora

Sapone

Iganga

Lambarene

Navrongo

Pooled

Hospital admissions: ITT

GMZ2 Rabies

Vaccine

VE

95%CI)


Banfora

Sapone

Iganga

Lambarene

Navrongo

Pooled


Deaths: ITT

GMZ2 Rabies

Vaccine

VE

95%CI)


Banfora

Sapone

Iganga

Lambarene

Navrongo

Pooled


Prevalence of anaemia on Day 252:

Hb<5 Hb<7 Mean Hb

GMZ2 Rabies

vaccine

VE

95%CI)

GMZ2 Rabies

vaccine

VE

95%CI)

GMZ2 Rabies

vaccine

Difference

95%CI)

Banfora

Sapone

Iganga

Lambarene

Navrongo

Pooled

Prevalence of asexual parasitaemia on days 84, 365 and 588.

Day84 Day365 Day588

GMZ2 Rabies

vaccine

VE

95%CI)

GMZ2 Rabies

vaccine

VE

95%CI)

GMZ2 Rabies

vaccine

VE

95%CI)

Banfora

Sapone

Iganga

Lambarene

Navrongo

Pooled

Prevalence of gametocyte carriage days 84, 365 and 588.

Day84 Day365 Day588

GMZ2 Rabies

vaccine

VE

95%CI)

GMZ2 Rabies

vaccine

VE

95%CI)

GMZ2 Rabies

vaccine

VE

95%CI)

Banfora

Sapone

Iganga

Lambarene

Navrongo

Pooled

Density asexual parasitaemia on days 84, 365 and 588. Arithmetic means.

Day84 Day365 Day588

GMZ2 Rabies

vaccine

VE

95%CI)

GMZ2 Rabies

vaccine

VE

95%CI)

GMZ2 Rabies

vaccine

VE

95%CI)

Banfora

Sapone

Iganga

Lambarene

Navrongo

Pooled

Density of gametocyte carriage days 84, 365 and 588.Arithmetic means.

Day84 Day365 Day588

GMZ2 Rabies

vaccine

VE

95%CI)

GMZ2 Rabies

Vaccine

VE

95%CI)

GMZ2 Rabies

vaccine

VE

95%CI)

Banfora

Sapone

Iganga


Lambarene

Navrongo

Pooled

Surrogates of protection: If there is evidence that the vaccine has a protective effect, further analysis to try to

understand which immune responses are associated with vaccine-induced protection will be required. To

evaluate the association between antibody titres and efficacy, malaria incidence measured at the 6 month

endpoint will be related to vaccine-specific immune responses measured post-dose 3. It is necessary to show

a) immune response is associated with malaria incidence in the control group, and in the malaria vaccine

group, and b) that the relationship is the same in both groups, and then to show that the difference in malaria

incidence between vaccine and control groups is explained by the immunological marker (Prentice, 1989; Qin

et al., 2007). Using the ATP population, each immunological response will be included in a Cox model, with the

intervention group as a covariate, to see to what extent the immunological variable explains the difference in

malaria incidence between the vaccine groups. This will be done for each immunological variable in turn using

immunological variables measured prior to and immediately post dose 3 and malaria incidence at 6 months.

Table: Hazard ratio (95%CI) from Cox models to investigate correlates of protection:

Variable Model 1: Model 2: Model 3:

Immune variables

Vaccine group

Covariates

[Technical notes:

1. It is common to consider the child is not at risk for a period of 2 to 4 weeks after each treatment (depending

on the drugs used for treatment), and to subtract this time from the time at risk, thereby giving an estimate of

vaccine efficacy as if treatment had no preventive effect. Since treatment effects do occur, this practice slightly

overestimates the actual effect of the vaccine.

2. When a child is treated for malaria, if an effective drug is used they would be expected to be free of

parasitaemia and symptoms after 2-3 days. Symptoms can recur days or weeks after the initial episode if

parasites are not completely eliminated. We need to define when recurring symptoms will be considered to be

a distinct episode. A common practice is to assume that symptoms that occur with 3 or 4 weeks of the initial

episode are most likely to be relapses and these are considered part of the initial episode. The efficacy is then

interpreted as the reduction in the number of disease episodes due to new infections, rather than the

reduction in the number of illness episodes. If a period of 7 days is used, each period of up to 1 week of illness

that meets the case definition is counted as a separate event. The protocol specified 14 days but a case could

be made for 7 days.

3. Vaccine efficacy (VE) means the percentage reduction in the number of malaria episodes during a specified

period of time. The statistical estimate using Cox regression should closely approximate the simple percent

reduction in number of cases. When there is no loss to follow-up (all children followed for the same length of

time to the end of the study), and the number of children in each group is the same, the hazard ratio (HR) from

Cox regression is exactly equal to the number of episodes in the vaccine group divided by the number of

episodes in the control group so 100x(1-HR) is the vaccine efficacy i.e. the percentage reduction in the number

of episodes. This is true even if efficacy varies with time, so the approach does not assume proportional

hazards. When there is loss to follow-up, there is an adjustment to allow for the duration of follow-up but the

interpretation is the same. Due to seasonality, if VE is calculated directly from the incidence rates in the two

groups, the estimate may be slightly different from the Cox estimate, because using the crude incidence rates

assumes incidence is constant and this introduces a slight bias when there is loss to follow-up.

Pooling over sites: The stratified Cox model allows the incidence and the pattern of seasonality to differ

between sites, when efficacy is the same in all sites this is the preferred method, but when efficacy varies

(interaction with site) it can be better to estimate overall efficacy by estimating efficacy separately in each site

by fitting a Cox model to the data for each site, and then combine the resulting estimates (Mehrotra et al.

2012). As an additional exploratory analysis this method will be used to produce an overall estimate of

efficacy.

ITT and ATP analyses : ITT gives the fairest assessment in the sense that it includes all subjects who were

randomized, but it includes periods of surveillance during which individuals have not been fully vaccinated.

ATP analysis, limited to those that were fully vaccinated, is intended to better reflect the efficacy of the full


course of vaccination, but may be influenced by bias due to exclusion of some subjects who were randomized.

These distinctions are well recognized, so it is not necessary for use to specify which is primary, both can be

presented. In general excluding from ATP has to be done judiciously if we exclude for every minor protocol

violation we may end up with small and unrepresentative sample, many minor violations are unrelated to

efficacy.

4. For each dose there was a stagger of some weeks between the first and last child to be vaccinated at each

site; using time from dose, instead of calendar date, as the time scale therefore introduces a slight extra

variability in incidence due to the seasonal pattern of malaria. Differences in the pattern of seasonality

between sites are allowed for by considering sites as strata in the Cox model, giving equal coefficients across

strata but with a baseline hazard unique to each stratum.

5. For malaria intervention studies the use of a case definition with a high cut-off parasite density is usually

recommended in order to have a highly specific case definition, excluding cases with low parasites densities in

whom malaria-like symptoms may be due to another illness. For a vaccine that works by reducing parasite

density rather than preventing infection, this approach may lead to bias because the vaccine could increase

number of people with parasite densities below the cut-off, compared to the control group, some of these will

have clinical malaria but would be excluded from the case definition (Smith, 2007). It is therefore necessary to

use a range of cut-off values, or alternatively to include all episodes with any parasitaemia and adjust for low

specificity. A probabilistic definition of efficacy could be used but this has a disadvantage that the estimated

efficacy does not correspond to a number of actual malaria cases prevented.

6. Parasitaemia is analysed in terms of the arithmetic mean rather than the geometric mean. For analysis of

gametocytes this is more appropriate, it is a better measure of average transmissibility than either the

prevalence or the geometric mean density among the positives. Geometric means are problematic because

they have to exclude all the negatives.]

Individual patient listings:

Normal ranges and AE gradings:

References:

Xu Y, Cheung YB, Lam E, Milligan PJM (2010) A simple approach to estimation of incidence rate difference.

American Journal of Epidemiology 172:334-343.

Cheung YB, Xu Ying, Tan Sze Huey, Cutts F, Milligan PJM (2010) Estimation of intervention effects using first or

multiple episodes in clinical trials: The Andersen-Gill model re-examined. Statistics in Medicine 29(3):328-336

Smith TA (2007) Measures of clinical malaria in field trials of interventions against Plasmodium falciparum.

Malaria Journal 6:53.

Prentice RL (1989) Surrogate endpoints in clinical trials: definition and operational criteria. Stat Med 1989;

8:431–40.

Qin L et al. (2007) A Framework for Assessing Immunological Correlates of Protection in Vaccine Trials

Journal of Infectious Diseases 196:1304–12

Senn S (1997) Statistical Issues in Drug Development. Wiley.

Mehrotra DV, Shu-Chih Sua, Xiaoming Lib (2012) An efficient alternative to the stratified

Cox model analysis.Statist. Med. 31:1849-1856

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