Supplementary appendix€¦ · Salomão, Anderson da Silva, Heloisa M. Espinola, Joane do Prado...

Supplementary appendixThis appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors.

Supplement to: Kallas EG, Precioso AR, Palacios R, et al. Safety and immunogenicity of the tetravalent, live-attenuated dengue vaccine Butantan-DV in adults in Brazil: a two-step, double-blind, randomised placebo-controlled phase 2 trial. Lancet Infect Dis 2020; published online Matrch 24. hhttps://doi.org/10.1016/S1473-3099(20)30023-2.

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APPENDIX

Safety and immunogenicity of Butantan-DV, a tetravalent, live

attenuated dengue vaccine evaluated in adults in an endemic region: a

randomized, placebo-controlled, double-blind, phase 2 clinical trial

Esper G. Kallás, Alexander Precioso, Ricardo Palacios, Beatriz Thomé, Patrícia E. Braga,

Tazio Vanni, Lúcia M. A. Campos, Lilian Ferrari Gabriella Mondini, Maria da Graça

Salomão, Anderson da Silva, Heloisa M. Espinola, Joane do Prado Santos, Cecilia L. S.

Santos, Maria do Carmo S. T. Timenetsky, João Luiz Miraglia , Neuza M. F. Gallina,

Daniela Weiskopf, Alessandro Sette, Raphaella Goulart, Rafael Tavares Salles, Alvino

Maestri, Adriana Maluf Elias Sallum, Sylvia Costa Lima Farhat, Neusa K. Sakita, Juliana

C. O. A. Ferreira, Cassia G. T. Silveira, Priscilla R. Costa, Isaias Raw, Stephen S.

Whitehead, Anna P. Durbin, Jorge Kalil.

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CONTENTS

Appendix A – Eligibility criteria for healthy adults, of both sexes, and between 18 and 59 years of age. To be included in the study participants had to meet all the inclusion criteria and could not fulfill any of the exclusion criteria. ................................................ 3

I. Inclusion criteria .................................................................................................... 3

II. Exclusion criteria ................................................................................................... 3

Appendix B ........................................................................................................................ 6

I. Severity classification ............................................................................................ 6 II. Causal relationship classification .......................................................................... 9

Appendix C ...................................................................................................................... 11

Figure 1: CD8+ T cell immune responses after vaccination. ..................................... 11

Table 1. Step A: Adverse Reactions after the first dose of Butantan-DV, TV003, or placebo ........................................................................................................................ 12

Table 2. Rash characteristics in participants from StepA and in all Butantan-DV recipients, after the first dose. ..................................................................................... 13 Table 3. Multivalent immune response after the first in Step A and all Butantan-DV recipients. .................................................................................................................... 14

Table 4. Frequency of vaccine rash according to the multivalent immune response in Step A and in all Butantan-DV recipients, after the first dose.................................... 15

Table 5. Step A: Frequency of seroconversion after first (S1) and second (S2) doses. .................................................................................................................................... 16

Table 6. Step A: Geometric mean titers following the first and second doses. .......... 17

Appendix D ..................................................................................................................... 18 Statistical Analysis Plan for the study “A Phase II, stepwise, randomized, double-blind, controlled clinical trial to evaluate the safety and immunogenicity of the lyophilized formulation of the Dengue 1,2,3,4 (attenuated) vaccine among healthy adults” ......................................................................................................................... 18

Data Sharing Statements ............................................................................................. 19

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Appendix A – Eligibility criteria for healthy adults, of both sexes, and between 18 and 59 years of age. To be included in the study participants had to meet all the inclusion criteria and could not fulfill any of the exclusion criteria.

I. Inclusion criteria

Potential participants will be included in the study only if they fulfill all criteria described below:

a. To be a healthy adult, of both sexes, and between 18 and 59 years of age;

b. To be available during the entire study period, which will be of approximately five years;

c. To demonstrate the intention to participate in the study, documented by the signature of the free informed consent form (FICF);

d. For females: volunteers with childbearing potential should be willing to use birth control methods until their last safety evaluation visit, which corresponds to the S12 visit for Step A, and the S6 visit for Step B. All female volunteers will be considered as having childbearing potential, except those that have a documented history of hysterectomy, tubal ligation or have reached menopause (12 months of amenorrhea after the last menstrual period). The volunteers who declare themselves as not having childbearing potential, because of sexual abstinence or non-reproductive sexual practices, until their last safety evaluation visit, which corresponds to the S12 visit for Step A, and the S6 visit for Step B, will not need to use birth control methods.

II. Exclusion criteria

Potential participants who fulfill any of the criteria described below will not be included in the study:

a. For females: Pregnancy (confirmed by a β-hCG positive test) or breastfeeding;

b. Evidence of active neurological, heart, lung, liver or kidney disease according to clinical history, physical examination and/or laboratory tests;

c. Diseases that impair the immune system, including: diabetes, cancer (except basal cell carcinoma) and autoimmune diseases;

d. Behavioral, cognitive or psychiatric diseases that in the opinion of the principal investigator or of his medical officer, could affect the ability of the potential participant to understand and collaborate with the requirements of the study protocol;

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e. Values of absolute neutrophil count, alanine aminotransferase (ALT) or serum creatinine equal to or greater than Grade 1, as defined in this Protocol;

f. Any use considered abusive of alcohol or drugs in the 12 months preceding the inclusion in the study (V1 visit) that caused medical, professional or family problems, as indicated by the medical history;

g. History of severe allergic reaction or anaphylaxis;

h. Diagnosis of asthma with a history of hospitalization due to the illness in the last six months prior to the inclusion in the study (V1 visit);

i. Suspected or confirmed fever within 72 hours prior to the vaccination or axillary temperature higher than 37.8°C on the day of the vaccination (V1 visit);

j. Screening or confirmatory test positive for HIV-1;

k. Screening or confirmatory test positive for hepatitis C virus (HCV);

l. Positive test for the hepatitis B virus surface antigen (HBsAg), or for the antibody against the hepatitis B virus "core" antigen (anti-HBc) alone;

m. Use of corticosteroids (excluding topical or nasal) or other immunosuppressive drugs during the 42 days prior to the inclusion in the study (V1 visit). It will be considered as immunosuppressive a dose of corticosteroids equivalent to ≥10 mg prednisone per day for more than 14 days;

n. Use of anticoagulant medication;

o. To have been vaccinated with a live virus vaccine within the last 28 days or with an inactivated virus vaccine within the last 14 days prior to the inclusion in the study (V1 visit), or to have any immunization scheduled for the first 42 days after the inclusion in the study (V1 visit);

p. History of asplenia;

q. To have received blood products in the last six months prior to study inclusion (V1 visit), including transfusions or immunoglobulin, or to have scheduled the administration of blood products or immunoglobulin during the first 42 days after vaccination (V1 visit);

r. Use of any investigational product within 42 days prior to the inclusion in the study (V1 visit), or to have scheduled to receive it after the inclusion in the study (V1 visit);

s. Participation in another clinical trial within the six months preceding the inclusion in the study (V1 visit);

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t. To deny permission for the storage of biological material for future research as defined in the FICF;

u. Any additional condition which, in the opinion of the principal investigator or his medical officer, could jeopardize the safety or the rights of a potential participant, or that could prevent her/him from complying with this protocol.

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Appendix B

I. Severity classification

Based on the guide “Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials” from the US Food and Drug Administration (USFDA).1,2

Classification of the severity of the solicited clinical adverse events.

Adverse Event Grade 1 Grade 2 Grade 3 Grade 4

Pain at the injection site of the investigational product

Does not interfere

with daily activities

Repeated use of non-narcotic

analgesic >24 hours OR

Mild interference with daily activities

Any use of a narcotic

analgesic OR

Prevent daily activities

Emergency department

visit* OR

Hospitalization

Erythema at the injection site of the investigational product †

25 – 50 mm 51 – 100 mm >100 mm

Necrosis OR

Exfoliative dermatitis

Swelling at the injection site of the investigational product †

25 – 50 mm

51 – 100 mm OR


>100 mm Necrosis

Induration at the injection site of the investigational product †

25 – 50 mm

51 – 100 mm OR


>100 mm Necrosis

Pruritus at the injection site of the investigational product

Does not interfere





visit* OR

Hospitalization

Nausea

Does not interfere


OR 1 to 2

episodes in 24 hours


OR More than 2


Prevent daily activities, it requires IV hydration


visit* OR

Hospitalization OR

Hypovolemic shock

Vomiting

Does not interfere


OR 1 to 2



OR More than 2


Prevent daily activities, it requires IV hydration


visit* OR

Hospitalization OR

Hypovolemic shock

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Adverse Event Grade 1 Grade 2 Grade 3 Grade 4

Fever 37.8 – 38.4°C 38.5 – 38.9°C 39.0 – 40.0°C >40°C

Chills

Slight cold sensation;

chills, teeth chatter

Moderate chills in the entire body, it requires the use of

narcotics

Serious or prolonged, no

response to narcotics

-----

Headache

Does not interfere






analgesic OR



visit* OR

Hospitalization

Retro-orbital pain

Does not interfere






analgesic OR



visit* OR

Hospitalization

Photophobia

Does not interfere





visit* OR

Hospitalization

Fatigue

Does not interfere





visit* OR

Hospitalization

Myalgia

Does not interfere





visit* OR

Hospitalization

Arthralgia

Does not interfere





visit* OR

Hospitalization

Viral skin eruption (viral rash) ‡

Present, but asymptomatic

Symptomatic (pruritus/pain), but mild interference


Symptomatic, prevent daily

activities


visit* OR

Hospitalization * It requires 12 hours or more of admission to a ward or emergency department for the management of the adverse event. † The value recorded should be measured at the largest diameter and as a continuous variable. ‡ Specify whether the skin rash is located in any region of the body or if it is widespread.

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Classification of the severity of the laboratory adverse events.

Adverse event Grade 1 Grade 2 Grade 3 Grade 4 Hemoglobin decrease compared to baseline - gm/dL

0.1 – 1.5 1.6 – 2.0 2.1 – 5.0 >5.0

Hemoglobin (women) - gm/dL 11.0 – 12.0* 9.5 – 10.9 8.0 – 9.4 <8.0

Hemoglobin (men) - gm/dL 12.5 – 13.5* 10.5 – 12.4 8.5 – 10.4 <8.5

Leukocytosis – cell/mm3 10,800†– 15,000

15,001 – 20,000

20,001 – 25,000 >25,000

Leukopenia – cell/mm3 2,500 – 3,500* 1,500 – 2,499 1,000 – 1,499 <1,000 Lymphopenia – cell/mm3 750 – 1,000* 500 – 749 250 – 499 <250 Neutropenia – cell/mm3 1,000 – 1,499* 500 – 999 300-499 <300 Eosinophilia – cell/mm3 650† – 1,500 1,501 – 5,000 >5,000 Hypereosinophilia Platelets Decreased – cell/mm3

125,000 – 140,000*

100,000 – 124,000

25,000 – 99,000 <25,000

Prothrombin time (PT) – increase by factor

1.0 – 1.10 x ULN

1.11 – 1.20 x ULN

1.21 – 1.25 x ULN >1.25 ULN

Partial thromboplastin time (PTT) – increase by factor

1.0 – 1.2 x ULN

1.21 – 1.4 x ULN

1.41 – 1.5 x ULN >1.5 x ULN

Proteinuria Trace 1+ 2+ Hospitalization or dialysis

Glycosuria Trace 1+ 2+ Hospitalization for hyperglycemia

Hematuria (red blood cells per high power field) 1† – 10 11 – 50 >50 and/or

gross blood

Hospitalization or packed red blood

cells (PRBC) transfusion

Urea – mg/dL 49.3† – 55.7 55.8 – 66.4 >66.4 Requires dialysis

Creatinine – mg/dL 1.5† – 1.7 1.8 – 2.0 2.1 – 2.5 >2.5 or requires dialysis

CPK – mg/dL 1.25 – 1.5 x ULN

1.6 – 3.0 x ULN

3.1 –10 x ULN >10 x ULN

Alkaline phosphate – increase by factor

1.1 – 2.0 x ULN

2.1 – 3.0 x ULN

3.1 – 10 x ULN >10 x ULN

ALT – increase by factor 1.1 – 2.5 x ULN

2.6 – 5.0 x ULN

5.1 – 10 x ULN >10 x ULN

AST – increase by factor 1.1 – 2.5 x ULN

2.6 – 5.0 x ULN

5.1 – 10 x ULN >10 x ULN

Bilirubin – when accompanied by any increase in ALT or AST – increase by factor

1.1 – 1.25 x ULN

1.26 – 1.5 x ULN

1.51 – 1.75 x ULN >1.75 x ULN

Bilirubin – when ALT and AST are normal – increase by factor

1.1 – 1.5 x ULN

1.6 – 2.0 x ULN

2.0 – 3.0 x ULN >3.0 x ULN

ILN: inferior limit of the normal range.ULN: upper limit of the normal range. * If the ILN is less than the upper limit of Grade 1, the ILN will be used as an upper limit to Grade 1.† If the ULN is greater than the lower limit to Grade 1, the ULN will be used as a lower limit to Grade 1.According to criteria NIC/NIH NCI3

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II. Causal relationship classification

All adverse events had their causal relationship to the investigational product classified according to the adapted classification of the "Uppsala Monitoring Centre" of the World4.

Classification of the causal relationship of the adverse events.

A reasonable causal relationship A causal relationship NOT reasonable

The Adverse Event is considered an Adverse Reaction The Adverse Event cannot be considered an Adverse Reaction

Certain Probable Possible Unlikely Not related A clinical event, including a laboratory test abnormality (abnormal value) with a plausible temporal relationship to the administration of the intervention;

A clinical event, including a laboratory test abnormality (abnormal value) with a reasonable temporal relationship to the administration of the intervention;

A clinical event, including a laboratory test abnormality (abnormal value) with a reasonable temporal relationship to the administration of the intervention;

A clinical event, including a laboratory test abnormality (abnormal value) that due to the timing of the administration of the intervention has an unlikely relationship, but not impossible;

A clinical event, including a laboratory test abnormality (abnormal value) that due to the timing of the administration of the intervention has no relationship;

It cannot be explained by a concurrent disease or other intervention or medication; The event must be definitive pharmacologically or phenomenologically (i.e., is an objective and specific disorder or a pharmacologically recognized phenomenon);

It is unlikely to be attributed to a concomitant disease or other medication or intervention;

It can also be explained by a concurrent disease or other medication or interventions;

Another disease or another medication provides a plausible explanation.

Another disease or another medication provides a plausible explanation.

The response to an interruption or withdrawal is plausible (pharmacologically, pathologically);

The response to the interruption or withdrawal is clinically reasonable;

There is a lack of information or lack of clarity about the withdrawal or interruption of treatment.

A satisfactory rechallenge procedure if necessary.

A rechallenge procedure is not necessary.

References

1. CBER/USFDA/USDHHS Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials

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[Internet]. Silver Spring: US Food and Drug Administration; 2007 [cited 2011 Set 29].Available at: http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/ucm091977.pdf

2. ICH Clinical Safety Data management: Definitions and Standards for Expedited Reporting [Internet]. Genebra: International Conference on Harmonisation of Technical Requirements ror Registration of Pharmaceuticals for Human Use; 1994 [cited 2012 Oct 15]. E2A. Available at: http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E2A/Step4/E2A_Guideline.pdf

3. NIC/NIH NCI Common Terminology Criteria for Adverse Events (CTCAE) [Internet]. Available at: http://evs.nci.nih.gov/ftp1/CTCAE/About.html.

4. UMC/WHO The use of the WHO-UMC system for standardised case causality assessment [Internet]. Uppsala:The Uppsala Monitoring Centre. Available at: http://www.who-umc.org/Graphics/24734.pdf.

http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/ucm091977.pdf

http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/ucm091977.pdf

http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E2A/Step4/E2A_Guideline.pdf

http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E2A/Step4/E2A_Guideline.pdf

http://evs.nci.nih.gov/ftp1/CTCAE/About.html

http://www.who-umc.org/Graphics/24734.pdf

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Appendix C

Figure 1: CD8+ T cell immune responses after vaccination.

A. 15 DENV-naïve volunteers were evaluated for antigen-specific CD8+ T cell responses by flow cytometry after stimulation with DENV-derived peptide pool after first and second TV003 doses. Responses were documented in all, but one, vaccine, compared to none in the placebo group; after the second shot, most vaccinees maintained detectable antigen-specific immune responses; only one placebo recipient developed detectable immune responses after confirmed DENV1 natural infection documented before the second vaccine shot. (B) 94% (35 of 37) of DENV-naïve and DENV-exposed participants who received one shot of Butantan-DV mounted antigen-specific CD8+ T cell immune responses at Day 91 after vaccination, as opposed to 13% (2 of 18) of placebo recipients.

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Table 1. Step A: Adverse Reactions after the first dose of Butantan-DV, TV003, or placebo

Adverse Reactions

Step A But-DV Placebo TV003

p* (n=19) (n=10) (n=17) n (%) n (%) n (%)

Solicited laboratorial adverse reactions ALT increase 4 (21.1) 0 (0.0) 3 (17.6) 0.305 Hemoglobin# 2 (10.5) 2 (20.0) 3 (17.6) 0.749 Leukopenia 5 (26.3) 0 (0.0) 3 (17.6) 0.206 Neutropenia 5 (26.3) 0 (0.0) 2 (1.8) 0.152 Lymphopenia 1 (5.3) 0 (0.0) 1 (5.9) 0.745 Thrombocytopenia 0 (0.0) 0 (0.0) 0 (0.0) NP Lymphocytosis 0 (0.0) 0 (0.0) 0 (0.0) NP Leukocytosis 0 (0.0) 0 (0.0) 0 (0.0) NP Monocytopenia 1 (5.3) 0 (0.0) 1 (5.9) 0.745 Neutrophilia 0 (0.0) 0 (0.0) 0 (0.0) NP Solicited local adverse reactions Pain 0 (0.0) 2 (20.0) 2 (11.8) 0.164 Erythema 0 (0.0) 0 (0.0) 0 (0.0) NP Swelling 0 (0.0) 0 (0.0) 0 (0.0) NP Pruritus (local) 0 (0.0) 0 (0.0) 1 (5.9) 0.418 Induration 0 (0.0) 0 (0.0) 0 (0.0) NP Solicited systemic adverse reactions Rash 16 (84.2) 0 (0.0) 13 (76.5) <0.001 Headache 10 (62.5) 2 (20.0) 6 (35.3) 0.213 Myalgia 3 (15.8) 0 (0.0) 1 (5.9) 0.313 Retro-orbital pain 2 (10.5) 1 (10.0) 1 (5.9) 0.873 Arthralgia 0 (0.0) 0 (0.0) 2 (11.8) 0.168 Nausea 0 (0.0) 1 (10.0) 0 (0.0) 0.159 Fatigue 0 (0.0) 0 (0.0) 0 (0.0) NP Pyrexia 0 (0.0) 0 (0.0) 0 (0.0) NP Photophobia 0 (0.0) 0 (0.0) 1 (5.9) 0.418 Vomiting 0 (0.0) 0 (0.0) 0 (0.0) NP Chills 0 (0.0) 0 (0.0) 0 (0.0) NP Unsolicited adverse reactions## Administration site macule 1 (5.3) 0 (0.0) 0 (0.0) 0.484 Anhedonia 0 (0.0) 0 (0.0) 1 (5.9) 0.418 Asthenia 1 (5.3) 0 (0.0) 2 (11.8) 0.469 Decreased appetite 1 (5.3) 0 (0.0) 1 (5.9) 0.745 Odynophagia 1 (5.3) 0 (0.0) 0 (0.0) 0.484 Palatal edema 1 (5.3) 0 (0.0) 0 (0.0) 0.484 Somnolence 0 (0.0) 0 (0.0) 1 (5.9) 0.418 Vessel puncture site pruritus 0 (0.0) 0 (0.0) 1 (5.9) 0.418 Pruritus (systemic) 1 (5.3) 0 (0.0) 1 (5.9) 0.745 * Chi-square test # change from baseline value ## Reactions with overall incidence ≥2% NP: not possible to calculate

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Table 2. Rash characteristics in participants from StepA and in all Butantan-DV recipients, after the first dose.

VARIABLES Step A Butantan-DV

Placebo p Butantan-DV TV003 p DENV-naïve DENV-exposed

Participants with Rash (AR) 16 13 63 51 5

Onset (days) 0.859* 11 (6 - 18) 11 (5 - 19) 14 (4 - 22) 0.740#

median (min-max) 10.6 (6 - 18) 10 (7 - 13)

Duration (days) 0.870* 7 (1- 21) 5 (0 - 33) 11 (1 - 13) 0.571#

median (min-max) 9 (1 - 16) 8 (6 -12)

n (% )of rash Grade 1 14 (87.5) 12 (92.3) >0.999** 56 (88.9) 40 (78.4) 5 (100) 0.189##

* Mann-Whitney test ** Fisher´s exact test # Kruskal-Wallis test ## Chi-square test

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Table 3. Multivalent immune response after the first in Step A and all Butantan-DV recipients.

Multivalent immune response

Step A

p*

Butantan-DV participants

p* Butantan-

DV TV003 DENV-naïve DENV–exposed

(n=16) (n=17) (n=85) (n=101)

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

Per protocol

Tetravalent 12 (75.0) 13 (76.5) >0.999 54 (63.5) 56 (55.5) 0.296

Trivalent 3 (18.8) 2 (11.7) 0.656 21 (24.7) 26 (25.7) >0.999

Bivalent - - NP 6 (7.1) 6 (5.9) 0.774

Monovalent - 1 (5.9) >0.999 2 (2.3) 8 (7.9) 0.113

No immune response 1 (6.2) 1 (5.9) >0.999 2 (2.3) 5 (5.0) 0.457

* Fisher's exact test

NP: not possible to calculate

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Table 4. Frequency of vaccine rash according to the multivalent immune response in Step A and in all Butantan-DV recipients, after the first dose.

Multivalent immune response

Step A

p*

Butantan-DV participants

p* Butantan-DV TV003 DENV-naïve DENV–exposed no

Rash/n %

Rash no

Rash/n %

Rash no

Rash/n %

Rash no

Rash/n %

Rash Per protocol

Tetravalent 10/12 83.3 11/13 84.6 >0.999 36/54 66.7 31/56 55.4 0.246

Trivalent 3/3 100 2/2 100 NP 13/21 61.9 13/26 50.0 0.557

Bivalent - - - - NP 2/6 33.3 2/6 33.3 >0.999

Monovalent - - 0/1 0.0 NP 1/2 50.0 0/8 0.0 0.200

No immune response 0/1 0.0 0/1 0.0 NP 0/2 0.0 0/5 0.0 NP

* Fisher's exact test

NP: not possible to calculate

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Table 5. Step A: Frequency of seroconversion after first (S1) and second (S2) doses.

SEROTYPE Butantan-DV (n=13) TV003 (n=14)

p* S1 S2 S1 S2

DENV1 12/13=92.3% 13/13=100% 13/14=92.9% 14/14=100% >0.999

DENV2 12/13=92.3% 13/13=100% 12/14=85.7% 12/14=85.7% >0.999

DENV3 11/13=84.6% 12/13=92.3% 12/14=85.7% 12/14=85.7% >0.999

DENV4 11/13=84.6% 12/13=92.3% 13/14=92.9% 14/14=100% >0.999

* Fisher's exact test for Butantan-DV´s Increase versus TV003´s Increase

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Table 6. Step A: Geometric mean titers following the first and second doses.

SEROTYPE Butantan-DV TV003

n GMT1 (95%CI)

GMT2 (95%CI) n GMT1

(95%CI%) GMT2

(95%CI) Per protocol

DENV1 9 628.84 (236.31 - 1673.43)

96.25 (25.38 - 365.03) 12 128.03

(69.45 - 236.00) 79.59

(32.67 - 193.88)

DENV2 9 575.75 (161.42 - 2053.54)

43.30 (20.49 - 91.52) 12 526.37

(135.14 - 2050.21) 46.16

(13.72 - 155.26)

DENV3 8 305.88 (62.85 - 1488.69)

45.34 (6.87 - 299.21) 11 123.63

(22.73 - 672.45) 15.29

(10.52 - 22.23)

DENV4 8 131.46 (67.04 - 257.77)

31.86 (9.16 - 110.77) 12 298.39

(139.86 - 636.62) 75.49

(36.18 - 123.41)

Intention to treat

DENV1 12 479.15 ( 201.89 - 1137.14)

160.61 (48.50 - 531.83) 13 163.82

(75.57 - 355.14) 112.25

(37.26 - 338.19)

DENV2 12 391.94 (103.91 - 1478.33)

28.41 (14.43 - 55.95) 12 526.37

(135.14 - 2050.21) 46.16

(13.72 - 155.26)

DENV3 11 289.95 (72.60 - 1158.05)

29.20 (11.45 - 74.48) 12 104.89

(21.832 - 503.92) 15.29

(10.52 - 22.22)

DENV4 11 116.91 (57.36 - 238.28)

47.71 (11.39 - 199.78) 13 306.59

(153.39 - 612.79) 77.71

(46.31 - 130.39)

GMT1: geometric mean titers for dose 1 GMT2: geometric mean titers for dose 2 CI: Confidence Interval

18

Appendix D

Statistical Analysis Plan for the study “A Phase II, stepwise, randomized, double-

blind, controlled clinical trial to evaluate the safety and immunogenicity of the

lyophilized formulation of the Dengue 1,2,3,4 (attenuated) vaccine among healthy

adults”

The data will be recorded directly by the research center in electronic CRFs

through the software OpenClinica® Enterprise 3.1, or a newer version (OpenClinica,

Waltham, MA, USA) locally installed on the sponsor's server. This is a validated system

and in use by several American and European institutions.

The database for the statistical analysis will be extracted directly from

OpenClinica® Enterprise software. The main statistical analysis software for the study

will be Stata 11.0 (StataCorp LP, College Station, TX, United States).

The objective of this phase II clinical trial is to determine the safety and

immunogenicity of the Butantan-DV in humans. This study, similarly to other Phase II

clinical trials is exploratory and not confirmatory; as such it has the objective of

describing the safety and immunogenicity profiles and not of formally testing statistical

hypotheses.

Descriptive approaches will be used to meet the defined objectives described in

the protocol, as well as formal statistical tests when appropriate. Results will be presented

in a tabular format, as well as graphically when appropriate.

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Data Sharing Statements

• The individual, identifiable data and the dictionary defining each field in the set

will not be shared with others;

• Summarized deidentified data will be provided in manuscript form. In addition,

some de-identified safety data will be provided to the centralized

database consortium sponsored by the NIAID/NIH and organized by the NIAID-

NIH and the International Institute of Vaccines (IVI) only. They will not be made

available publicly;

• The study protocol, statistical analysis plan and informed consent may be shared

with licensees of the TV003/TV005 candidate vaccines from the NIAID-NIH that

agreed to participate in the centralized database consortium sponsored by the

NIAID/NIH and organized by the NIAID-NIH and the International Institute of

Vaccines (IVI) only.

• Data will be made available publicly with the publication in the Lancet Infectious

Disease Journal if the manuscript is accepted by the Journal;

• Summarize data may be shared with the licensees of the TV003/TV005 candidate

vaccines from the NIAID-NIH that agreed to participate in the centralized

database consortium sponsored by the NIAID/NIH and organized by the NIAID-

NIH and the International Institute of Vaccines (IVI) only. The data sharing will

occur upon request and only be approved after the members of the consortium

sign the data access agreement. Furthermore, the data access will be limited to

members of the consortium only who may share the information with their

respective regulatory agencies.

Supplementary appendix€¦ · Salomão, Anderson da Silva, Heloisa M. Espinola, Joane do Prado...

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