Protocol Summary: Clinical efficacy and safety of a novel
tetravalent vaccine for Severe Dengue
December 9, 2015
Wendy Charles, MS
Miriam Estin
Isobelle Galeon, MD, MPH
Puujee Jambal, MPH
1. Background and setting:
Describe the disease or condition. Include basic epidemiology as appropriate.
Describe current treatment(s) or standard of care. Describe the new treatment or
intervention that will be evaluated in your study. If relevant, consider the following
topics from my previous instructions:
Background on the disease or condition that is being targeted
A global public health problem, dengue is currently the most rapidly spreading mosquito-borne viral
disease in the world. The incidence of dengue infections have increased 30-fold in the last 50 years
(WHO, 2009). From 2003 to 2013, the reported cases of dengue have increased by a factor of five
(WHO, 2009). An estimated 390 million dengue infections occur annually, approximately 75% of
which occur in the Southeast Asian and Western Pacific Regions, with case fatality rates ranging
from 1-5% (WHO, 2009).
Primary or secondary infections with any of the 4 distinct serotypes of the dengue virus (DEN-1 to 4), a small single-stranded RNA virus that belongs to the genus Flavivirus and the family Flaviviridae,
occur through the bites of infected Aedes mosquitoes (mostly Ae. aegypti). Infection with any one of
the 4 types of dengue virus results in a wide spectrum of illness ranging from asymptomatic or
subclinical disease to severe dengue, which manifests with plasma leakage, hemorrhagic
tendencies, and death.
The WHO traditionally classified symptomatic infections as undifferentiated fever, dengue fever (DF),
and dengue hemorrhagic fever (DHF). The latter category is further classified into four severity
grades, with grades III and IV comprising the dengue shock syndrome (DSS). However, this
classification has been recently revised to DF with or without warning signs for the development of
severe dengue, and severe dengue itself (WHO, 2009; McArthur, Sztein, & Edelman, 2013). Patients
with dengue and without warning signs present with fever plus any two of the relevant symptoms
(i.e., nausea/vomiting, rash, aches/ pains, leukopenia, and/or a positive tourniquet test) and can be
managed at home. Those diagnosed with warning signs fulfilled the above criteria plus one or more
symptoms/signs of abdominal pain/tenderness, persistent vomiting, clinical fluid accumulation,
mucosal bleeding, lethargy/restlessness, liver enlargement >2 cm, and/or laboratory testing showing
increased hematocrit (>20% above patient’s baseline value or with concurrent rapid decrease in
platelet count) and need to be managed in a hospital. Patients with severe dengue present with
severe plasma leakage (i.e. pulmonary edema), bleeding (i.e., melena), and organ involvement (i.e.,
impaired consciousness) and require emergency treatment. Although most dengue cases follow a
self-limiting non-severe course, a small number of patients progress to severe disease. In the
Americas, a DHF case fatality rate of 1.2% was reported between 2007-2009 (WHO, 2009). DHF
was only reported in 9 countries prior to 1970 (WHO, 2009). This number has subsequently
Page 1 of 14
increased to more than 4-fold and continues to rise.
Summary of previous studies in this area. What is known? What is the broader
context for the proposed trial?
Vaccination against the dengue virus is currently being explored to lessen the significant burden of
disease on the estimated 2.5 billion people who reside in over 100 endemic countries worldwide
(WHO, 2009). Since severe dengue tends to occur among young children due to their apparent
inability to effectively compensate for capillary leakage, several studies have been recently
conducted in this population.
A recent study (Capeding et al., 2014) demonstrated the clinical efficacy (vaccine efficacy: 60.8%;
hospitalization for virologically-confirmed disease: 67.2%) and safety of a tetravalent dengue vaccine
among 10,275 healthy children, ages 2-14 years old in 5 Asian Pacific countries.
A subsequent follow-up study (Villar et al., 2015) enrolled 20,869 healthy children, ages 9-16 years
old, from 5 Latin American countries similarly demonstrated 60.8%, 80.3%, 95.5%, and 91.7%
vaccine efficacy rates for dengue fever infection, hospitalization for virologically-confirmed disease
(VCD), and hospitalization for severe VCD after 1 and 3 doses, respectively.
Risk factors
Several risk factors that have been found to directly influence dengue severity include secondary
infection, age, ethnicity, and chronic diseases. Although primary dengue virus infection is believed to
induce lifelong protective immunity to the infecting serotype, and provide protection from clinical and
severe illness within 2-3 months and up to 2 years after infection with a different serotype,
respectively, no long-term cross-protective immunity has been demonstrated (WHO, 2009; Flipse &
Smit, 2015).
After the cross-protective period, the risk of developing more severe dengue rises with a secondary
infection with a heterotypic serotype and the time between the primary and the secondary infection. A
higher case fatality rate has been observed when infection with the DEN-1 virus was followed by
DEN- 2 infection after an interval of 20 years, compared to an interval of four years (WHO, 2009).
Although uncommon, a few severe cases with primary infection have also been reported and
primarily occur in infants who were born to dengue-immune mothers. Antibody-dependent
enhancement (ADE) is believed to be responsible for severe clinical syndrome that is observed in
secondary infections and among primarily infected infants. This results in the binding of nonneutralizing, cross-reactive antibodies acquired from a primary infection, or passively at birth, to the
surface of a heterologous infecting virus (WHO, 2009). This facilitates virus entry into Fc-receptorbearing cells resulting in more infected cells, a higher viral burden, a robust host immune response
(i.e., the release of inflammatory cytokines and mediators), capillary leakage and eventually, the
characteristic signs and symptoms of severe dengue infections. Once activated, cross-reactive
memory T cells also rapidly proliferate, express cytokines, and undergo apoptosis, all of which
influences the overall disease severity.
What is the key scientific and clinical question that needs to be addressed?
Given the previously presented facts about dengue vaccines and the severity of secondary
infections, we have decided to focus our study on investigating the efficacy of the tetravalent vaccine
in preventing severe dengue infections among adults with previous VCD. This focus is also pertinent
from a public health viewpoint with consideration of the burden on hospitals during endemic
transmission seasons and epidemic outbreaks. We aim to address the following relevant clinical
question:
Page 2 of 14
Does the administration of 3 doses of a recombinant, live, and attenuated tetravalent dengue vaccine
(CYD-TDW) in a 0-6-12 month schedule, lead to fewer hospitalizations secondary to severe dengue,
among healthy adults with at least one previous episode of virologically-confirmed disease (VCD)?
The intended indication for the new approach (treatment, intervention, or new test)
including: the disease or condition, the target population, the treatment or
intervention, and the desired outcome.
Approach: Vaccine intervention
Disease: Virologically-confirmed severe dengue disease, including Dengue Hemorrhagic Fever
(DHF) and Dengue Shock Syndrome (DSS)
Target population: Healthy adults who have had at least one episode of virologically-confirmed
disease (VCD) in Southeast Asia and the Western Pacific
Intervention: Recombinant, live, and attenuated tetravalent dengue vaccine that will be administered
in 3 doses, on a 0-6-12 month schedule
Desired outcome: Fewer confirmed hospitalizations for severe dengue
Summary of indication: Recombinant, live, attenuated tetravalent dengue vaccine intervention for the
reduction in severity of dengue fever in healthy adults who have had at least one episode of
virologically-confirmed disease in order to reduce the number of hospitalizations for severe dengue.
A description of where the proposed trial fits in the sequence of studies (e.g., the
phase of the trial).
This study will be a Phase III study to establish the clinical efficacy of the tetravalent dengue vaccine
and superiority over placebo. As additional considerations to justify Phase III status, earlier studies
were conducted on the vaccine to establish the dosing and toxicity, and this study involves very large
sample size where the study population closely reflects the target population.
Give one or two references that describe the setting, or are similar studies to the one
you are proposing.
Capeding MR, Tran NH, Hadinegoro SRS, Ismail HIHJ, Chotpitayasunondh T, Chua MN, Luong CQ,
Rusmil K, Wirawan DN, Nallusamy R, Pitisuttithum P, Thisyaforn U, Yoon I, van der Vliet D, Langevin
E, Laot T, Hutagalung Y, Frago C, Boaz M, Wartel A, Tornieporth NG, Saville M, Boukenooghe A,
and the CYD14 Study Group. Clinical efficacy and safety of a novel tetravalent dengue vaccine in
healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet.
2014; 384:1358-1365.
Flipse J, Smit JM. The complexity of a dengue vaccine: a review of the human antibody response.
PLoS Neglected Tropical Diseases. 2015; 9(6):1-18.
Hadinegoro SR, Arredondo-García JL, Capeding MR, Deseda C, Chotpitayasunondh T, Dietze R, et
al. Efficacy and Long-Term Safety of a Dengue Vaccine in Regions of Endemic Disease. New
England Journal of Medicine. 2015;373(13):1195-206.
McArthur MA, Sztein MB, Edelman R. Dengue vaccines: recent developments, ongoing challenges,
and current candidates. Expert Review of Vaccines. 2013; 12(8):933-953.
Villar L, Dayan GH, Arredondo-García JL, Rivera DM, Cunha R, Deseda C, Reynales H, Costa MS,
Morales-Ramirez JO, Carrasquilla G, Rey LC, Dietze R, Luz K, Rivas E, Montoya MCM, Supelano
Page 3 of 14
MC, Zambrano B, Langevin E, Boaz M, Tornieporth N, Saville M, Noriega F, CYD15 Study Group.
New England Journal of Medicine. 2015; 372(2):113-123.
WHO. Dengue guidelines for diagnosis, treatment, prevention, and control. Geneva: World Health
Organization; 2009.
2. Synopsis of proposed study:
(a) Study population: Outline eligibility and exclusion criteria
Inclusion criteria: This study will be conducted among healthy adults who are/have:
●
18-45 years old
●
Previous virologically-confirmed dengue (VCD)
●
Residing in urban areas and cities in the endemic provinces of Southeast Asia and the
Western Pacific regions
●
Have access to participating study field centers and hospitals for enrollment and followup
Exclusion criteria: We will exclude patients with:
●
Circulating antibodies to other flaviviruses (i.e., Yellow fever) to prevent cross-reactivity
●
Previously diagnosed/pre-existing immunocompromised states (i.e., autoimmune
diseases, HIV, cancer, diabetes mellitus)
●
Identified contraindications to the vaccine and its administration (i.e., with
hypersensitivity/adverse drug reactions to the vaccine)
●
In the absence of previous studies on the effect of the vaccine on nursing or pregnant
mothers, we will also exclude these women from this study.
Case Identification:
Severe cases of dengue will be identified clinically, based on the 2009 WHO criteria, and
confirmed by laboratory tests (viral nucleic acid detection by RT-PCR and ELISA). In this study, a
case is defined as an episode of symptomatic severe dengue accompanied by evidence of
severe plasma leakage, hemorrhage, and organ impairment, which was confirmed virologically by
means of enzyme-linked immunosorbent assay for dengue nonstructural protein 1 antigen,
dengue screening on quantitative reverse-transcriptase–polymerase-chain-reaction (RT-PCR)
assay, or serotype-specific RT-PCR assay. Physicians with specialized training in infectious
diseases and who are not involved in the patient’s clinical care will verify cases of severe dengue
for inclusion in the analysis.
(b) Study treatment(s): Describe study treatments. Use an appropriate level of detail.
Treatments:

Vaccine/Placebo:
o The tetravalent vaccine consists of four recombinant dengue vaccine viruses (CYD 1
through 4). The strains were be developed by Sanofi Pasteur by substituting genes
encoding the proteins of the yellow fever 17D vaccine virus with those from wild-type
dengue viruses. Vaccine formulations were combined into a single preparation containing
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5.0 log10 median cell-culture infectious doses (CCID50) per serotype and were formulated
as a powder and solvent (0.4% sodium chloride) for suspension (Villar, et. al., 2015).
o Placebo involves a 0.9% solution of sodium chloride.

Injection: The vaccine or placebo injections will be administered subcutaneously above the
deltoid.
Dosing: Either dengue vaccine or placebo will be administered in 3 doses, on a 0-6-12 month
schedule
(c) Study measurements: Define and describe the study measurements that are part of
the study. Usually there is a verbal description of each measurement and the
measurement method accompanied by a “visit table” showing the visit schedule
and measurements made at each visit.
The following measurements will be collected to confirm baseline status and assess for severe
dengue disease over the course of study participation. Refer to the Visit Table provided in Figure
1.
Blood samples:
Blood samples will be collected up to three times during study participation to test for dengue
non-structural protein 1 antigen (Capeding, et. al, 2014). If the test is positive, subjects will be
classified as having virologically-confirmed disease.
1. As part of routine participation, blood samples will be obtained from all subjects at months 0
and 13.
2. Subjects suspected of reinfection with acute febrile illness will be asked to come to the clinic
to provide blood samples for laboratory exams to confirm dengue infection (i.e., complete
blood count, RT-PCR, ELISA) and assess the severity of infection (i.e., liver function tests,
serum electrolytes, BUN and creatinine, bicarbonate or lactate, etc.). The goal is to obtain the
first sample within 5 days of acute symptoms and the second sample 7 – 14 days later.
Telephone/home follow-up:
1. During the Active Phase (through month 25), subjects will be contacted every week to collect
vaccine-related adverse events and to determine the emergence of dengue disease.
2. During the Hospital Phase (from Year 3 to Year 6), subjects will receive a phone or home visit
every 3 months until the end of the entire study period to collect adverse events and to
determine the emergence of dengue disease.
3. Each subject will be asked to immediately contact the study center/coordinator if he/she
develops acute high-grade fever of at least 3 days’ duration at any time after study enrollment
for possible dengue infection, as well as potential vaccine-related adverse events.
Questionnaire:
At screening, subjects will complete a questionnaire to collect information regarding their previous
medical and medication history (including past history of dengue infection).
Medical records:
Subjects will provide study coordinators with access to their medical records for post-vaccination
hospitalization due to severe dengue within the study period. Medical records will be reviewed:
1. At the time of post-vaccination hospitalization for suspected dengue virus.
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2. At the conclusion of each subject’s participation (due to withdrawal or completion) to
determine unreported hospitalizations for severe dengue, to confirm the diagnosis of severe
dengue, and to identify potential vaccine-related adverse events.
(d) Statistical design: Describe the general design. Describe the primary, secondary,
and exploratory endpoints for the trial. Propose and evaluate the sample size.
Briefly describe the statistical analysis plan:
Trial design: describe the type of design (e.g., double-blind placebo-controlled
randomized controlled trial).
The main objective of the trial is to determine long-term safety of the dengue vaccine and to verify
that vaccination reduces the risk of severe disease (measured by the incidence of hospitalization
for DHF/DSS) in adults in whom immunity has not been additionally boosted.
This study will be designed as a randomized (2:1), placebo-controlled, observer-masked, phase
III, multi-center, multi-country trial.
Study Timeline:
Active Phase: Vaccine efficiency and safety will be assessed during 25-month surveillance phase
(until 13 months after the third dose was administered).
Hospital Phase: Long-term vaccine safety will be assessed in the follow-up period (from year 3 to
year 6), using incidence of hospitalization for DHF/DSS.
The overall study timeline will be 6 years as depicted in Figure 1.
Figure 1
Study endpoints: List and describe primary endpoint, key secondary endpoints,
and exploratory endpoints. Note that these should also be listed in the “visit
table”.
Primary outcomes:
Annual incidence rate of hospitalization for dengue (DHS/DSS) calculated as the number of
cases divided by the total number of participants. The relative risk of hospitalization for VCD will
Page 6 of 14
be calculated as the ratio of the annual incidence in vaccine group to that in control group, which
is expressed as Vaccine Efficacy (1 − RR).
Secondary outcomes:
1. ICU admission for Severe Dengue
2. Mortality rates due to Severe Dengue
3. All-cause mortality
Visit Table:
The duration of each subject’s participation in the trial will be approximately 6 years. As noted
earlier, the duration of the Active Phase for each subject is expected to be 25 months (12 months
of surveillance after Dose 3), and the duration of the Hospital Phase for each subject is expected
to be 47 months. Each subject is expected to have a total of 11 Visits during the trial as outlined
in the Visit Table below:
Visit Table
V02
V03
V04
V05
V06
V07
V08
V09
V10
V11
D0
D28
D180
V03+28d
D365
V05+28
d
Last
Vacc.
+13
months
Last
Vacc.
+24
months
Last
Vacc.
+36
months
Last
Vacc.
+48
months
Last
Vacc.
+60
months
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Dose 1
x
x
x
x
x
x
Dose 2
x
Immunogenicity Test (Subset)
Informed Consent
Inclusion/Exclusion Criteria
Urine Pregnancy Test
Contraindications
Physical Exam
Clinical Exam and Temp
History of Dengue or YF
Infection/Vaccination
Concomitant therapy
Randomization
BL (Dengue Ab)
Vaccination
Memory Aid (MA)
*Detection of Dengue
Cases & Hospitalizations
Serious Adverse Events
V01
Immunogenicity Test (Subset)
Visit Number (V)
Timelines
x
x
x
x
x
x
Dose 3
x
x
x
Throughout the entire trial
Throughout the entire trial
Adopted from Villar L et al. N Engl J Med 2015;372:113-123
For V08, V09, V10, and V11, all subjects will be contacted by phone/home visit and will be
reminded to come to the study center in case of febrile illness or in case of an adverse event. A
clinical examination will be conducted at the Investigator’s discretion and the blood sample will be
taken for dengue neutralizing Ab.
There will be weekly contacts during Active Phase and at least every 3 months during Hospital
Phase. Detection of virologically-confirmed dengue cases, hospitalization related to DHS/DSS,
and any serious adverse events, including mortality will be monitored throughout the entire study
period.
Sample size evaluation: Use methods from class to propose and evaluate the
sample size for your study.

Type of trial: e.g., superiority, non-inferiority, equivalence.
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Superiority trial. We hypothesize that vaccination will reduce the incidence of
hospitalization for DHF/DSS

Parameterization of outcome space including primary endpoint, probability
model, functional, contrast, statement of statistical hypotheses.
Primary endpoint: Hospitalization within 5 years after receiving 3 doses of vaccine
expressed as Vaccine efficiency (VE).
Annual incidence rate of hospitalization for dengue (DHS/DSS) calculated as the number
of cases divided by the total number of participants. The relative risk of hospitalization for
VCD will be calculated as the ratio of the annual incidence in vaccine group to that in
control group, which is expressed as Vaccine Efficacy (1 − RR).
Probability model: Incidence rates (Hospitalizations for VCD) follows Poisson dist.
Functional: Mean rate
𝜃0 = 𝜆0 → 𝑡ℎ𝑒 𝑖𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 𝑜𝑓 𝑉𝐶𝐷 𝑝𝑒𝑟 𝑝𝑒𝑟𝑠𝑜𝑛 − 𝑦𝑒𝑎𝑟 𝑖𝑛 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝
𝜃1 = 𝜆1 → 𝑡ℎ𝑒 𝑖𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 𝑜𝑓 𝑉𝐶𝐷 𝑝𝑒𝑟 𝑝𝑒𝑟𝑠𝑜𝑛 − 𝑦𝑒𝑎𝑟 𝑖𝑛 𝑉𝑎𝑐𝑐𝑖𝑛𝑒 𝑔𝑟𝑜𝑢𝑝
Ν0 → 𝑡ℎ𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑒𝑟𝑠𝑜𝑛 − 𝑦𝑒𝑎𝑟𝑠 𝑖𝑛 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝
Ν1 → 𝑡ℎ𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑒𝑟𝑠𝑜𝑛 − 𝑦𝑒𝑎𝑟𝑠 𝑖𝑛 𝑉𝑎𝑐𝑐𝑖𝑛𝑒 𝑔𝑟𝑜𝑢𝑝
Contrast: Rate Ratio
𝜃=
𝜃1
𝑤ℎ𝑒𝑟𝑒 𝜃1 → 𝑖𝑠 𝑡ℎ𝑒 𝑖𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 𝑜𝑓 𝑉𝐶𝐷 𝑖𝑛 𝑉𝑎𝑐𝑐𝑖𝑛𝑒 𝑔𝑟𝑜𝑢𝑝
𝜃0
𝜃0 → 𝑖𝑠 𝑡ℎ𝑒 𝑖𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 𝑜𝑓 𝑉𝐶𝐷 𝑖𝑛 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝
𝜽 < 𝟏. 𝟎 𝒊𝒏𝒅𝒊𝒄𝒂𝒕𝒆𝒔 𝒃𝒆𝒏𝒆𝒇𝒊𝒕
Hypothesis:
In terms of Vaccine Efficiency (VE):
𝐻0 : 𝑉𝐸 ≤ 70% 𝑚𝑒𝑎𝑛𝑠 𝑅𝑅 ≥ 0.3 ( ℎ𝑎𝑟𝑚)
𝐻+ : 𝑉𝐸 > 70% 𝑚𝑒𝑎𝑛 𝑅𝑅 < 0.3 ( 𝑏𝑒𝑛𝑒𝑓𝑖𝑡)
𝑉𝐸 = 1 − 𝑅𝑅 = 1 −
𝜃1 𝜃0 − 𝜃1
=
𝜃0
𝜃0
In terms of Rate Ratio:
𝑯𝟎 : 𝜃 ≥ 𝜃∅ ⇒ 𝜃∅ = 1.0 ⇒ 𝜽 ≥ 𝟏. 𝟎
𝑖𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 𝑜𝑓 𝑉𝐶𝐷 𝑤𝑖𝑙𝑙 𝑏𝑒 𝑠𝑎𝑚𝑒 𝑖𝑛 𝐶𝑜𝑛𝑡 & 𝑉𝑎𝑐 𝑔𝑟𝑜𝑢𝑝𝑠
𝑯+ : 𝜃 < 𝜃+ ⇒ 𝜃+ = 0.3 ⇒ 𝜽 < 𝟎. 𝟑
𝑎𝑡 𝑙𝑒𝑎𝑠𝑡 70% 𝑟𝑒𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑜𝑓 𝑉𝐶𝐷 𝑖𝑛 𝑉𝑎𝑐𝑐𝑖𝑛𝑒 𝑔𝑟𝑜𝑢𝑝

Statistical design of the fixed-sample trial including choice of information
and evaluation of the sample size.
Evaluation of the sample size:
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Information needed to calculate to discriminate the above hypotheses:
𝜃 ≥ 1.0 𝑎𝑛𝑑 𝜃 < 0.3 𝑓𝑜𝑟 90% 𝑝𝑜𝑤𝑒𝑟:
From the formula sheet:
𝑆𝐸 [log 𝑒 (
̂1
𝜃
1
1
+
)] = √
𝜃0
𝜃0 𝑁0 2𝜃1 𝑁0
Without 𝑁0 or 𝑁1 , use the Variance for Rate Ratio from the formula sheet:
𝑉=
1
1
+
𝑤ℎ𝑒𝑟𝑒 𝜃0 = 1.0 𝑎𝑛𝑑 𝜃1 = 0.3; 𝑟 = 2 (1: 2)
𝜃0 𝑟𝜃1
𝑉=
1
1
1
+
=1+
= 2.67
1.0 2 × 0.3
0.6
Number of events (D) in each arm:
2
𝑧𝛼 + 𝑧𝛽
𝐷=(
) ×𝑉
𝜃+ − 𝜃∅
1.96 + 1.28 2
=(
) × 2.67
log 𝑒 (0.3)
3.24 2
=(
) × 2.67
−1.2039
= 7.24 × 2.67 = 19.3
𝐷 = 19 → 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑒𝑣𝑒𝑛𝑡𝑠 𝑖𝑛 𝑃𝑙𝑎𝑐𝑒𝑏𝑜 𝑔𝑟𝑜𝑢𝑝
𝑁1 = 2 × 𝑁0 𝑡ℎ𝑒𝑛 𝐷 = 19 × 2 = 38 𝑓𝑜𝑟 𝑉𝑎𝑐𝑐𝑖𝑛𝑒 𝑔𝑟𝑜𝑢𝑝
19 + 38 = 𝟓𝟕 𝒆𝒗𝒆𝒏𝒕𝒔
How many person-years will it take to observe 57 dengue cases?
Incidence rate of dengue =64 per 10,000 person-years (i.e., 0.64 per 100):
19
19
=
× 10,000 = 2, 969 𝑝𝑒𝑟𝑠𝑜𝑛 − 𝑦𝑒𝑎𝑟𝑠 𝑖𝑛 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝
64
64
10,000
2, 969 + 594 (20% 𝑑𝑟𝑜𝑝𝑜𝑢𝑡) = 𝟑, 𝟓𝟔𝟑 𝒔𝒖𝒃𝒋𝒆𝒄𝒕𝒔 𝒇𝒐𝒓 𝑪𝒐𝒏𝒕𝒓𝒐𝒍 𝒈𝒓𝒐𝒖𝒑
38
38
=
× 10,000 = 5, 938 𝑝𝑒𝑟𝑠𝑜𝑛 − 𝑦𝑒𝑎𝑟𝑠 𝑖𝑛 𝑉𝑎𝑐𝑐𝑖𝑛𝑒 𝑔𝑟𝑜𝑢𝑝
64
64
10,000
5, 938 + 1, 188 (20% 𝑑𝑟𝑜𝑝𝑜𝑢𝑡) = 𝟕, 𝟏𝟐𝟔 𝒔𝒖𝒃𝒋𝒆𝒄𝒕𝒔 𝒇𝒐𝒓 𝑽𝒂𝒄𝒄𝒊𝒏𝒆 𝒈𝒓𝒐𝒖𝒑
Total of 10,689 subjects
Conclusions:
Assuming an incidence rate of VCD of 64 per 10,000, an overall drop-out rate of 20%,
and the true VE of 70% after 3 doses, a total of 57 dengue cases or 10,689 subjects
Page 9 of 14
are expected to provide >90% power (i.e., per-protocol analysis (PPA) including all
subjects who had no protocol deviations).
Reality scenario:
In reality, for a full-analysis (FA) assuming an overall vaccine efficiency of 55% after at
least one 1 dose:
𝑉=
1
1
+
𝑤ℎ𝑒𝑟𝑒 𝜃0 = 1.0 𝑎𝑛𝑑 𝜃1 = 0.45; 𝑟 = 2 (1: 2)
𝜃0 𝑟𝜃1
𝑉=
1
1
1
+
=1+
= 2.11
1.0 2 × 0.45
0.9
Number of events (D) in each arm:
2
𝑧𝛼 + 𝑧𝛽
𝐷=(
) ×𝑉
𝜃+ − 𝜃∅
1.96 + 1.28 2
=(
) × 2.11
log 𝑒 (0.45)
3.24 2
=(
) × 2.11
−0.7985
= 16.46 × 2.11 = 35
𝐷 = 35 → 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑒𝑣𝑒𝑛𝑡𝑠 𝑖𝑛 𝑃𝑙𝑎𝑐𝑒𝑏𝑜 𝑔𝑟𝑜𝑢𝑝
𝑁1 = 2 × 𝑁0 𝑡ℎ𝑒𝑛 𝐷 = 35 × 2 = 70 𝑓𝑜𝑟 𝑉𝑎𝑐𝑐𝑖𝑛𝑒 𝑔𝑟𝑜𝑢𝑝
𝑻𝒐𝒕𝒂𝒍 𝒐𝒇 𝟑𝟓 + 𝟕𝟎 = 𝟏𝟎𝟓 𝒆𝒗𝒆𝒏𝒕𝒔
How many person-years will it take to observe 105 (35+70) dengue cases?
Incidence rate of dengue =64 per 10,000 person-years (i.e., 0.64 per 100):
35
35
=
× 10,000 = 5, 469 𝑝𝑒𝑟𝑠𝑜𝑛 − 𝑦𝑒𝑎𝑟𝑠 𝑖𝑛 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝
64
64
10,000
5, 469 + 1, 094 (20% 𝑑𝑟𝑜𝑝𝑜𝑢𝑡) = 𝟔, 𝟓𝟗𝟑 𝒔𝒖𝒃𝒋𝒆𝒄𝒕𝒔 𝒇𝒐𝒓 𝑪𝒐𝒏𝒕𝒓𝒐𝒍 𝒈𝒓𝒐𝒖𝒑
70
70
=
× 10,000 = 10, 938 𝑝𝑒𝑟𝑠𝑜𝑛 − 𝑦𝑒𝑎𝑟𝑠 𝑖𝑛 𝑉𝑎𝑐𝑐𝑖𝑛𝑒 𝑔𝑟𝑜𝑢𝑝
64
64
10,000
10, 938 + 2, 188 (20% 𝑑𝑟𝑜𝑝𝑜𝑢𝑡) = 𝟏𝟑, 𝟏𝟐𝟔 𝒔𝒖𝒃𝒋𝒆𝒄𝒕𝒔 𝒇𝒐𝒓 𝑽𝒂𝒄𝒄𝒊𝒏𝒆 𝒈𝒓𝒐𝒖𝒑
Total of 19,719 subjects
Conclusions:
Assuming an incidence rate of VCD of 64 per 10,000, an overall drop-out rate of 20%,
and the true VE of 55% after at least 1 dose, a total of 105 dengue cases or 19, 719
subjects are expected to provide >90% power (i.e., full-protocol analysis (FA) including
all subjects who received at least one injection).
For PPA, we expect to observe 57 dengue cases and recruit 10,689 subjects.
Page 10 of 14
For FA, we expect to observe 105 dengue cases and recruit 19,719 subjects.
Choice of information:
With the best possible scenario (PPA) and selecting the information to discriminate the
hypothesis 𝜃 ≥ 1.0 𝑎𝑛𝑑 𝜃 < 0.3:
1 1
1
1
𝑆𝐸 = √ + ≈ √ +
= 0.2809
𝐷 𝐷
19 38
𝐶𝑉 = 𝑒 −1.96×0.2809 = 𝑒 −0.550564 = 0.58
95% 𝐶𝐼 𝑎𝑡 𝐶𝑟𝑖𝑡𝑖𝑐𝑎𝑙 𝑉𝑎𝑙𝑢𝑒 = 𝑒 (−0.550564±1.96×0.2809)
= (𝑒 −1.101128 ; 𝑒 0 )
= (0.33; 1)
Potential conclusions:
Statistical significant benefit
If 𝜃̂ < 0.58 ⇒ 𝑟𝑒𝑗𝑒𝑐𝑡 𝜃 ≥ 1.0
Not significance:
If 𝜃̂ ≥ 0.58 ⇒ 𝑟𝑒𝑗𝑒𝑐𝑡 𝜃 ≥ 0.3
Analysis plan: Describe the primary analysis (e.g., a 2-sample t-test of the
difference between means). The secondary analyses usually use the same
methods as the primary analysis but with a different outcome variable.
Statistical Analysis Plan:
We will utilize “intent to treat” for our primary analyses in order to minimize the effects of loss-tofollow-up and noncompliance. In addition, we will secondarily perform “per protocol” or “completecase” analyses in order to get additional information about the true efficacy of the vaccine as it is
recommended. However, because the “per protocol” analysis may introduce bias into our study
(for example, over-reporting the rate of severe dengue or overestimating the effect of the
vaccine), so we will be cautious in interpreting those findings.
Incidence and Efficacy Calculations:
We will calculate annual incidence rates and 95% confidence intervals for all dengue infections
(measured by virologically-confirmed infection by all serotypes) in each group, as well as for
hospitalizations due to severe dengue (virologically-confirmed DSS or DHF, defined according to
WHO criteria). Incidence rates will be calculated as percentage of all participants who got at least
one dose of the vaccine, who developed one or more qualifying event per 12 month period.
Vaccine efficacy against infection and against hospitalization due to severe dengue will be
expressed as 1 minus the relative risk, which we will calculate as the ratio of the annual incidence
in the vaccine group to that in the control group.
In order to control for loss to follow-up or different durations of follow-up after the first dose of the
vaccine, incidence density will also be calculated. This will be expressed as the cumulative
Page 11 of 14
person-time at risk (number of cases per 100 person-years at risk) for each group. In this
analysis, vaccine efficacy will be expressed as a hazard ratio.
Vaccine Efficacy Over Time:
In order to identify any waning in vaccine efficacy over time, percent disease-free survival
(without infection or without severe dengue) will be calculated during the trial. This data will be
displayed as a Kaplan-Meier plot, and differences between the groups will be assessed using the
Kaplan-Meier Estimate.
Multivariate Analyses:
Multivariate Cox proportional hazards regression analyses will be also be conducted in order to
control for any effects due to variables for which we stratified our blocked randomization. These
include: treatment group, gender, hospital district, and age (with age as a continuous
variable).Next, subgroup analyses (significance set to p=0.1) will be performed to detect
interactions between hospitalization for severe dengue and other factors, including region, viral
serotype, and baseline antibody titer (as a continuous variable). A Forest Plot will be generated
based on these findings, and post-hoc statistical tests for interaction will be performed
(significance at p = 0.1). Any statistically significant findings will be assessed for clinical
relevance.
Secondary Outcomes:
Vaccine efficacy against specific secondary outcomes (ICU admission for DHF/DSS, and
mortality rates due to DHF/DSS, and all-cause mortality) will also be assessed as previously
described, and expressed as relative risk, calculated as the ratio of the annual incidence of these
outcomes in the vaccine group to that in the control group.
3. Study implementation and conduct: Describe key elements of how the study will be
implemented. Depending on your trial you might want to consider:
Recruitment and retention plans: How will you identify potential participants and
make sure that they complete all outcome measurements.
Recruitment: We will identify health clinics within the targeted (largely) urban communities that will
facilitate subject recruitment for this study. Participating clinics and hospital emergency rooms will be
asked to provide a list of adult patients who were diagnosed and therapeutically managed for at least
one episode of VCD. Study coordinators will subsequently contact these individuals for potential
study inclusion and to schedule a screening visit.
Prospective subjects will be asked to review a consent form approved by the local ethics review
board prior to study enrollment. A member of the study team will then discuss the details of
participation with each prospective subject (and his/her family when consistent with local customs) in
a language understandable to them. No study procedures will be performed until each subject (and
when appropriate, the subject’s family) provides affirmation of the agreement to participate.
Retention plans: Because subjects will be followed up to 6 years, we expect a certain level of
dropouts. To increase retention:
●
Study staff will receive detailed training about interaction and communication styles that help
engage subjects in the study process and help subjects feel valued.
●
Subjects will be provided with a visit calendar with the clinic’s contact information to facilitate
advance planning for study visits.
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●
Subjects will receive a reminder phone call before each appointment and allow for timely
rescheduling, if needed.
●
Subjects can speak with a study doctor when they have questions about symptoms or
participation.
●
Subjects will be offered convenient timing of appointments, including evening hours and weekend
appointments. Some participating clinics will offer home follow-up visits, when possible.
●
Subjects will be provided with a modest stipend payment after completion of each study visit or
follow-up visit. (We would consult each local ethics board for recommendation of stipend amount
(if any) that would not cause undue enticement for that community.)
●
Study staff will provide initial and ongoing education about the importance of the trial and the
value of ongoing participation.
Randomization procedures: Describe how participants will be randomized, and how
the study will mask the randomization. Attach an appendix with the randomization
table for your trial.
Subjects will be randomized in a 2:1 ratio to receive three doses of tetravalent vaccine or placebo.
Because this is a multi-site trial, participating clinics will use an interactive voice-response or webresponse system to assign subjects to groups.
Randomization will be performed with computer-generated blocks of ten, stratified according to
treatment group, gender, hospital district, and age group (18-27 years, 28-36 years, and 37-45
years).
Procedures for blinding: Describe how the study will be blinded.
Blinded: Investigators, participants, and sponsor will be blinded to study group assignments. Also, an
independent data monitoring committee will perform a blinded review of each case of suspected
reinfection to assess the severity of infection.
Not blinded: Due to the nature of injection preparation procedures, the injections of vaccine or
placebo will be prepared and administered by staff members who are aware of study group
assignments. These staff members will not be involved in study assessments.
Procedures for minimizing missing data: Describe procedures that will be used to
prevent missing data.
To minimize missing data from a study design standpoint, participating clinics will:
●
Educate each subject about the goals of the study—especially the importance of recognizing and
reporting suspected infection with dengue virus. Each subject will be provided with a
thermometer, symptom card, temperature recording card, and clinic contact card to report
possible infection.
●
Follow-up with each subject by phone or in person to obtain adverse events, ask about possible
infection, and emphasize the importance to reporting suspected re-infection.
●
Ask each subject to provide contact information for a friend or family member who would be
knowledgeable about the subject’s condition so that health information could be obtained when
the subject cannot be reached.
Page 13 of 14
●
If subjects request to discontinue future vaccinations, they will be asked to continue with the
follow-up visits and to allow study-related access to their medical records.
●
Review medical records from the clinic and local hospital(s) to try to obtain information related to
treatment or hospitalization.
To address missing data from a statistical analysis standpoint, both “intent to treat” and “complete
case” (“per protocol”) analyses will be performed. In addition, incidence density as well as cumulative
incidence will be calculated, and Kaplan-Meier plots will be used to track vaccine efficacy over time.
Page 14 of 14