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Clinical Evaluation of Pandemic Live
Attenuated Influenza Vaccine (PLAIV)
Candidate Strain A/17/CA/2009/38 (H1N1) in
Healthy Thais
Punnee Pitisuttithum - MBBS, DTM&H,FRCPT
Faculty of Tropical Medicine , Mahidol University
August 17,2011, Salaya,MU
WHO-Major approaches to increasing supplies of pandemic influenza vaccine
Develop regional and national plans for seasonal influenza vaccination programmes
Build a new production facilities in developing and/or industrialized countries
Explore formulations of influenza vaccine other than those commonly used for seasonal vaccination
3
Live Attenuated Influenza Virus Vaccine
4
5Russian Donor strain
Potential advantages of LAIVs• no down-stream processing required (harvested
vaccine is simply packaged);• high yield (20-50 doses of monovalent vaccine
per an egg);• needle-free delivery (administration is via an
intra-nasal spray), which may facilitate administration in resource-poor settings;
• induction of a broad immune response including mucosal, systemic and cell-mediated responses (in contrast parenterally administered inactivated vaccines do not induce mucasal immunity);
• induction of cross-reactive immune responses;
การพั�ฒนาวั�คซี�น-ขขึ้ �น
ทะเบี�ยนการทดสอบีระยะท�� 3 ประส�ทธิ�ผล
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การศึ กษาในส�ตวั&ทดลอง (ควัามปลอดภั�ยและภั!ม�ต#านทาน)
การศึ กษาในห้#องปฏิ�บี�ต�การ
-ข
ในการที่�ผ่�านแต่�ละระยะจะต่�องผ่�านเกณฑ์�ที่�ก�าหนดไว้�ต่��งแต่�เร มต่�น
History of LAIV Development in Thailand
• 2004 outbreak of H5N1 in Thailand • 2005 Indonesia stop sending flu viruses to WHO• 2006 Serious discussion on equitable sharing of benefits
from sharing viruses• 2007 WHO/HQ started supporting developing countries to
build up capacity to produce Flu vaccine in 6 countries, including Thailand
• 2008 Thailand success in producing first seasonal IIV at 2 doses per egg
• May 2009 Sublicensing agreement on LAIV with WHO based on Russian Technology from IEM, St Petersberg
• 16th July 2009 received the H1N1 (2009) pre-Master seeds from Russia, through WHO support and start MS, WS, vaccine viruses
• August 12th first PLAIV vaccine concentrate was harvested
• August 25th first PLAIV clinical lot filled and tested
10
H1N1 2009 LAIV
A/California/07/2009 (H1N1).A/Leningrad/134/17/57 (H2N2) X
A/17/California/2009/38 (H1N1) vaccine
Pre master seed
Facility for GMP production of clinical trial lot certified by WHO
Initial ObstraclesFor production
• Less yield, low dose than expected • SPF eggs-imported-German, US -not available in Thailand-Stabilizer??
PLAIV vaccine intranasal route
15
• Nasal applicator-imported• Need to be available locally
Characterization of candidate vaccines Vaccine Production-identity(genotype,
phenotype);toxicity (mice,guinea pig ferret) Potency-immune response (mice , ferret) Stability Batch release and Independent Laboratory
evaluation Standards and Reference Materials
H1N1 2009 GPO vaccine
Pre master seed from Russia
master seed
Working seed
Virus concentrates
Bulk vaccine Vaccine product
Identity- genotypic, phenotypic
Toxicities-mice,guinea pigImmuninty and attenuation in animal model
Each step required
วั�คซี�นไขึ้#ห้วั�ดให้ญ่.ชน�ดเช0�อเป1นอ.อนฤทธิ�3• ม�ควัามไวัต.ออ"ณห้ภั!ม�
• เจร�ญ่เต�บีโตได#ในท��เย7น• อ.อนฤทธิ�3
การศึ กษาในส�ตวั&ทดลอง1. ควัามปลอดภั�ย
–เช0�อไม.ก.อให้#เก�ดโรค–เช0�อย�งคงม�ค"ณสมบี�ต�
• เต�บีโตได#ด�ในท��เย7น ~ 33oC
•ตายเม0�ออ"ณห้ภั!ม� > 38oC
•ย�งคงอ.อนฤทธิ�32. ระด�บีภั!ม�ต#านทาน
PRE–CLINICAL STUDY (1)
INFECTIVITY: 7.5–8.5 lg EID50/0.2 ml
IDENTITY (CONFIRMATION OF GENOME FORMULA): 6:2
GENETIC STABILITY (CONFIRMATION OF STABILITY OF MUTATIONS IN THE GENOME OF VACCINE STRAIN RESPONSIBLE FOR
THE ATTENUATION): by Faculty of Science ,MU
GMP facility for CLINICAL lot production is at Silapakorn U
PRE–CLINICAL STUDY (2)
GUENEA PIGS ( by GPO, Fac. Vet, MU) AND FERRETS STUDY (Amsterdam): for Safe & immunogenicityImmune response –at Fac.Medicine,Siriraj Hosp,MU
ATTENUATION (CONFIRMATION OF THE ts/ca/att PHENOTYPE): ts/ca (IN HENS’ EGG). att (IN MICE). TOX STUDY: SAFE FOR MICE removal of adventitious agents by Faculty of Medicine, Siriraj,MU
WILD TYPE AND COLD–ADAPTEDVIRUSES IN FERRETS
Lungs Nasal turbinates
Wild type virus Vaccine strain
lg E
ID5
0/m
L/g
r
1 2 3 4 5 6 7 8 9 100
2
4
6
8attenuated
non–attenuated
ส8าห้ร�บีวั�คซี�นห้วั�ด การทดสอบีระยะท�� 3
ประส�ทธิ�ผล การทดสอบีระยะท�� 2 ควัามปลอดภั�ยและภั!ม�ค"#มก�นการทดสอบีระยะท�� 1 ควัาม
ปลอดภั�ยในมน"ษย&การศึ กษาในส�ตวั&ทดลอง (ควัามปลอดภั�ยและภั!ม�ต#านทาน)
การศึ กษาในห้#องปฏิ�บี�ต�การ
Phase I/II Safety and Immunogenicity of
Pandemic Live Attenuated Influenza
Vaccine (PLAIV) Candidate Strain
A/17/CA/2009/38 (H1N1) in Healthy Thais
Punnee Pitisuttithum - MBBS, DTM&H,FRCPT
Faculty of Tropical Medicine , Mahidol University
Objectives
Overall Primary objectives (Part A, Part B)• To evaluate safety and reactogenicity of PLAIV
manufactured by GPO, Thailand• To evaluate humoral immune response of the above
vaccine after intranasal application by using HAI test, micro neutralization assays
• To determine the vaccine induced local IgA response by ELISA
Secondary objectives (Part A)• To assess shedding and stability of the viral strain by
using PCR method.
Part A:• The study is a double blind randomized study
involving 24 participants with the main objective of assessing safety-tolerability and optimal immune response of the newly manufactured PLAIV candidate strain A/17/CA/2009/38 (H1N1).
• It is study of two different inoculums sizes of candidate vaccines (5.0-6.5 log10 EID50 or 6.6-7.5 log10 EID50) and given two doses 21 days apart.
Pandemic Live Attenuated Influenza Vaccine (PLAIV) Candidate Strain A/17/CA/2009/38 (H1N1) in 5% sucrose (0.5ml intranasally)
Vaccine :
Part A• 24 Volunteers• Divided into 2 groups, 12 volunteers each group• Group 1 received 5.8 log 10 EID50• Group 2 received 6.9 log 10 EID50
Group Placebo 5.0 - 6.5 log 10 EID50A/17/CA/2009/38 (H1N1)
6.6 - 7.5 log 10 EID50
A/17/CA/2009/38 (H1N1)
N
1 3 9 0 12
2 3 0 9 12
Vaccine : Placebo = 3:1
Part B:• The study is a double blind randomized study
involving 324 participants with the main objective of assessing immune responses and safety of the newly manufactured PLAIV candidate strain A/17/CA/2009/38 (H1N1).
• Using the PLAIV of 6.5-7.5 log10 EID50 and given in intranasally two doses 21 days apart.
Group >12-18 years
>18-49 years >49 years
Vaccine 81 81 81
Placebo 27 27 27
Table 1.Number of participants in 3 stratified age groups
Total =108 each arm
Activities (Part A)
Immunization D1,D21 (Two doses)
Safety follow up for 7 days after
each immunization and
D21,D42,D60
Nasal swab D2,3,5,7
Nasal wash D1,21,42,D60
Blood drawn D1,D21,D42,D60
Other systemic reactions will be assessed in 4 scales as follow:
0 - no No symptoms
1 -mild Ill-defined symptoms
2 –moderate Symptoms, affecting normal daily activity
3 -severe Symptoms markedly affecting normal daily
activity and needed medication or clinic visit or activity limit
• Data & Safety Monitoring Board-Chair-MU
(Advised by WHO regional office and TDR) • Monitoring by WHO-TDR• Audit-initial audited by WHO-TDR from
Roch• Safety monitoring team appointed by
sponsor-Chaired by Prof. Dr. Apornpirom
88 screen (14 re-screened)
24
12 12
11 12
Low dose 5.8 (EID 50) High dose 6.9 (EID 50)
High titre HAI 16/78 (20.5%)Abnormal CxR 7/88Other abnormalities
1st Immunization
2nd Immunization
Abnormal CXR (re-read)
Delayed Immunization (URI)-PCR for seasonal and pandemic flu-negative
(2)
Results
Reactogenicity –Part A
• One had fever from the high dose group(38 oc)
• Few reported cough ,scratchy throat and myalgia from both groups and also the placebos.
• Reactogenicity declined after second immunization
Immunological Measurements (Siriraj Hospital)
Specific antibodies analysis includes specific anti-influenza antibodies to the vaccine strain (HAI test, ELISA, micro-neutralization assay).
HAI assay Sera were pre-treated with receptor
destroying enzyme and were tested for hemagglutinin-inhibition (HI) antibodies using standard procedures and whole influenza A and B viruses homologous to the vaccine strains in a standard micro-titer assay
40 Samples were sent to NSBC –WHO reference lab: different in HI titre, the NSBC results in relatively higher titer.
Micro-neutralization assay
mNT was performed in MDCK cell monolayer using the local isolate, A/Thailand/104/2009(H1N1) as well as A/CA/07/2009(H1N1) as the test viruses.
sIgA in the nasal wash
- Nasal wash specimens were tested for influenza virus specific IgA antibodies using an enzyme-linked immunosorbent assay (ELISA).
• Nasal swab samples were collected on D2, 3, 5 and 7 after each vaccination.
• Viral shedding in nasal swab samples was determined by real time RT-PCR for M gene using CDC protocol for the 2009 pandemic influenza virus.
• Sensitivity of detection is limited to 10-100 copies/reaction.
• RNP was used as the house keeping gene.
Conclusion• LAIV Candidate Strain A/17/CA/2009/38 (H1N1) appeared to be safe in small number of healthy adults
• Viral shedding rate was low since it was
found only in two cases at D2 after first vaccination.
• In term of immune responses , it is difficult to
conclude since the sample size was small and there
may be other immune responses that should be
measured other than HAI and MN for LAIV vaccine for example local IgA or cell mediated immunity
• DSMB recommendation:• No safety concern• Continue as planned to children and
middle age group
Phase I/II Safety and Immunogenicity of Pandemic Live Attenuated Influenza Vaccine (PLAIV) Candidate Strain A/17/CA/2009/38
(H1N1) in Healthy Thais
Punnee Pitisuttithum MB,BS, DTM&H,FRCP(T)
Faculty of Tropical Medicine , Mahidol University
17-18 Feb 2011.Geneva
Part B
Group >12 – 18 years
>18 – 49 years
>49 years
Vaccine 81 81 81
Placebo 27 27 27
Vaccine: Placebo = 3:1 (108 each group)
Study Design It is a double blind randomized study using the
6.6-7.5 log10 EID50 dose (stabilizer is similar to Medimmune) 324 participants (243 vaccines and 81
placebos) will be enrolled
Activities
• Immunization D1,D21 vaccine using stabilizer which is similar to Med Immune LAIV vac
• Safety follow up for 7 days after each immunization,D21,42,60
• Nasal wash D1.D21,42,60 (ONLY 40 IN ADULT GROUP)
• Blood drawn D1,21,42,60
Group I (Age > 12-18)
Total Screen = 404 cases(male= 195, female= 209) ; Rescreen = 6 cases
Total enroll = 110 cases (male= 49, female= 61)
Not eligible = 294 cases (HI Titer=234, *Others = 60)
Group II (Age ≥ 18-49)
Total Screen = 267 cases(male= 101, female= 166)
Total enroll = 110 cases (male= 44, female= 66)
Not eligible = 157 cases (HI Titer=82, *Others = 75)
Screen Group II replace Group I
Total Screen = 21 cases(male= 1, female= 20); Rescreen = 10 cases
Total enroll = 10 cases (male= 0, female= 10)
Not eligible = 11 cases (HI Titer=5, *Others = 6)
Group III (Age ≥ 49)
Total Screen = 356 cases(male= 63, female= 293) ; Rescreen = 24 cases
Total enroll = 133 cases (male= 18, female= 115)
Not eligible = 223 cases (HI Titer=56, *Others = 167)
*(Others ; Abnormal CXR , Anemia, HBsAg +ve, HCV +ve, chronic disease, High AST, ALT, CPK , Abnormal urine, etc…)
GPO FLU VACCINE -01 Part B
Total Screen = 1048 casesTotal enroll = 363 casesNot eligible = 685 cases
Rescreen = 40 cases
40% reported AE suspected to be related to the vaccine and equally distributed in all three age groups
not related to treatment related to treatment related to treatment (cause permanent discontinuation
of treatment)
related to treatment (cause temporary discontinuation
of treatment)
0
10
20
30
40
50
60
70
80
90
100Total Age group
>12-18 Yr
>18-49 Yr
> 49 Yrtotal
Blinded data only those reported AEsare shown in the graph
• Common AE suspected to be related to the treatment were rhinorrhea (8%)
throat irritation (5%), nasopharyngitis (8%) cough(4%) and upper respiratory tract infection (1%)
• The majority were mild,19% reported as moderate and only 2% reported severe in intensity
• There is no serious adverse event (SAE) reported in part B
Bad Taste in Mouth
Burning Sensation
Cough Nasal Blockage
Nausea Redness of Nose
Runny Nose
Scratchy Throat
Sore Throat
Stiffness of Nose
Vomit0
5
10
15
20
25
30
First Immunization N = 310 Second Immunization N = 276
* 27.5%
Comparison of Local Reaction Post Immunization(from total volunteers) majority were mild grade
Subj
ects
, %
10%
16%
5%
19%
11%14%
6%5.8%
1%
14%
29%
13%
21%
11%
27.5%
13%
1.8%
Comparison of Maximum Grade of Local Reaction at Post Immunization separate by age group
Total Age Group
>12-18 Yr >18-49 Yr > 49 Yr0
10
20
30
40
50
60
70
80
90
100
Grade 1
Grade 2
Grade 3
Total Age Group
>12-18 Yr >18-49 Yr > 49 Yr0
10
20
30
40
50
60
70
80
90
100
Grade 1
Grade 2
Grade 3
Mild
1st immunization
Sub
ject
s, %
2nd immunization
Sub
ject
s, %
Mild
Grade 2 Grade 30
5
10
15
20
25First Immunization N = 310 Second Immunization N = 310
>37.5-38.5 >38.5
Subj
ects
, %Comparison of Fever - Post Immunization(from total volunteers)
N=6%
N=2%
N=0.6%
0
5
10
15
20
25
30
First Immunization N = 310 Second Immunization N = 310
*23%
Comparison of Systemic Reaction Post Immunization(from total volunteers)
Subj
ects
, %
6%
7%
17%
11%
7% 7%
19%
7%0
9%10%
9%
6%
13%
0.03%
Comparison of Maximum Grade of Systemic Reaction at Post Immunization separate by age group (out of those who reported)
Total Age Group
>12-18 Yr >18-49 Yr > 49 Yr0
10
20
30
40
50
60
70
80
90
100
Grade 1
Grade 2
Grade 3
Su
bje
cts,
%
Total Age Group
>12-18 Yr >18-49 Yr > 49 Yr0
10
20
30
40
50
60
70
80
90
100Grade 1
Grade 2
Grade 3
1st immunization 2nd immunization
Sub
ject
s, %
Mild
Mild
Haemagglutination inhibition test (HAI Assay)
Total Age Group
Test virus: A/17/CA/2009/38(H1N1) – GPO vaccine strain
Day Vaccination status N
Number of subjects with HI antibody titer of
No. with Ab rising in folds
<10 10 20 40 80 160 2 4
1 Pre-IMM 266 247 19 - - - - - -
21Before 2nd
IMM260 202 34 16 4 3 1
4618%
197.3%
4221 D after 2nd
IMM 258 158 49 31 14 4 291
35%47
18.2%
6039 D after 2nd
IMM 260 151 43 39 20 5 210239%
6224%
Blinded data
Since V:P 3:1
• If assume, all seroconversion by HI were due to vaccinees
• >2 folds rise D21 D42 D60
24% 47% 52%• >4 fold rise 10% 24% 31%
Conclusion
Vaccine appeared to be safeAbout 54% reported AE suspected to be related The most common ones are
nasopharyngitis,rhinorrhoea,cough which were mild in intensity
No SAE reportedOnly one reported fever>38.5 0CAbout 25% reported scratchy throat was the most
common local reaction The most common reported systemic reaction was
headache , only 5%reported as moderate and above in intensity
Immune responses
• Form blinded data,46.2% developed >2 fold rise and 24.3% developed >4 fold rise of immune responses measured by either HAI, Micro-neutralization test or sIgA from nasal wash
Licensed for Pandemic use –July12,2011 for adult
Serum Institute of India
Acknowledgement
Mahidol UniversityFaculty of ScienceFaculty of Medicine ,Siriraj HospitalFaculty of Veterinary MedicineFaculty of Tropical Medicine
• Vaccine manufactured-GPO
Sponsor by Thai Health, WHO
Dr. Suwit Wibulphonprasert ,MOPHDr. Vichai Chokviwat,MOPHDr. Marie-Paule Kieny WHODr.Larisa Rodenko, University of St Petersberg , Russia
Thank you for your attention
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