Clinical evaluation of
the LID live attenuated
tetravalent dengue vaccine
Steve Whitehead, Dec 2010
Laboratory of Infectious Diseases, NIAID, NIH
What are the goals (and caveats) of dengue vaccination?
Most of the millions of
adults on the streets of
Vietnam are dengue
immune.
What are the goals (and caveats) of dengue vaccination?
Most of the millions of
adults on the streets of
Vietnam are dengue
immune.
How did they get that way?
Can we safely induce this
immunity in children?
What are the goals (and caveats) of dengue vaccination?
In endemic areas, dengue
immunity is most likely
acquired by sequential
infections, the majority of
which are asymptomatic.
This leads to neutralizing
antibody with broad
specificity to all 4 DENV
serotypes.
What are the goals (and caveats) of dengue vaccination?
Dose
(2 – 7 yr
interval)
Vaccine
serotype
Mean PRNT on indicated day
of secondary dose % sero-
conversionDay 0 Day 28 Day 42
1o DEN 4 20 214 193 88
2o DEN 1 < 10 312 264 100
In endemic areas, dengue
immunity is most likely
acquired by sequential
infections, the majority of
which are asymptomatic.
This leads to neutralizing
antibody with broad
specificity to all 4 DENV
serotypes.
homotypic
homotypic
What are the goals (and caveats) of dengue vaccination?
Dose
(2 – 7 yr
interval)
Vaccine
serotype
Mean PRNT on indicated day
of secondary dose % sero-
conversionDay 0 Day 28 Day 42
1o DEN 4 20 214 193 88
2o DEN 1 < 10 312 264 100
DEN 2 < 10 159 169 75
DEN 3 < 10 118 176 75
In endemic areas, dengue
immunity is most likely
acquired by sequential
infections, the majority of
which are asymptomatic.
This leads to neutralizing
antibody with broad
specificity to all 4 DENV
serotypes.
homotypic
homotypic
heterotypic
heterotypic
What are the goals (and caveats) of dengue vaccination?
But wait!
Dengue immunity
acquired by sequential
infection is not without
its problems.
Primary
infection
Secondary
infection
(different serotype)
Antibody
dependent
enhanced
disease
?
What are the goals (and caveats) of dengue vaccination?
Sequential administration of
monovalent vaccines would
not be safe in the endemic
setting.
Alternatives:
-- Administer a single vaccine
that is cross-protective
against all four serotypes
-- Simultaneously administer
four vaccines against each
dengue serotype
LID / NIAID Dengue Vaccine Goals:
Tetravalent: a) DEN1
b) DEN2
c) DEN3
d) DEN4
Safe: a) For vaccinee
b) For community
c) Genetically stable
Immunogenic: Significant response after 1 dose
Economical: Available to those most in need
homotypic antibody to each serotype
C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3’5’
∆30rDEN1∆30
rDEN4∆30
Attenuation strategies for dengue virus vaccine candidates
∆30 deletion mutation in 3’-UTR:
CTG G TA A A C AGAA C A GCC AG G G ||| AG-C A CGG A T-A C A A-T G-C C-GT*G C-GC-G T-AA-T T-AA G A-TA AT C-GC-G T*GG-C A-TT-A C-GC-GACTA G-C| G A-TG-CC-GC C-GG-C C-G T TA-T A-T C T AACG-C G*T C-G C AG-C A A T-AAT GGTTCT--3’G-C G G C || ||| A G
--GTAAAACCT-A G A TGTCGTCCTAGAGACC A T |||||| ||||GACACAAC AAACAGCA CTGG G
G A TATTGACG G AC AA TG AC AA A G AG-C G GC-G A A G-C G*TG-C A-TG-C C-GG-CC-GAC GC-GACTAC-GG AA AG-CAC-GT TC TT CG-CA-TG-CG-CG AA TA GG C T
∆30
C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3’5’
∆30rDEN1∆30
rDEN4∆30
C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3’5’
∆30 rDEN1/4∆30
rDEN2/4∆30
rDEN3/4∆30
Attenuation strategies for dengue virus vaccine candidates
30 deletion mutation in 3’-UTR:
Antigenic chimeras (rDEN4∆30 background):
prM and E region from wildtype DEN1, DEN2, or DEN3
C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3’5’
∆30rDEN1∆30
rDEN4∆30
C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3’5’
∆30 rDEN1/4∆30
rDEN2/4∆30
rDEN3/4∆30
Attenuation strategies for dengue virus vaccine candidates
30 deletion mutation in 3’-UTR:
Point mutations:
*C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3’5’
∆30rDEN4∆30-4995
rDEN4∆30-200,201
**
Antigenic chimeras (rDEN4∆30 background):
rDEN3∆30/31C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3’5’
∆30/31Additional 3’-UTR deletion mutations:
CTG G T
A A A C AGAA C A GCC AG G G ||| AG-C A CGG A T-A C A A-T G-C C-GT*G C-GC-G T-AA-T T-A
A G A-TA AT C-GC-G T*GG-C A-TT-A C-GC-GACTA G-C| G A-TG-CC-GC C-GG-C C-G T TA-T A-T C T AACG-C G*T C-G C AG-C A A T-AAT GGTTCT--3’G-C G G C || ||| A G
--GTAAAACCT-A G A TGTCGTCCTAGAGACC A T |||||| ||||GACACAAC AAACAGCA CTGG G
G A TATTGACG G AC AA TG AC AA A G AG-C G GC-G A A G-C G*TG-C A-TG-C C-GG-CC-GA
C GC-GACTAC-G
G AA AG-CAC-G
T TC TT CG-CA-TG-CG-C
G AA TA GG C T
∆30
∆31
Attenuation strategies for dengue virus vaccine candidates
C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3’5’
∆30
rDEN3-3’D4∆30
3’-UTR swap with rDEN4∆30:
DEN4
C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS55’ rDEN3
Attenuation strategies for dengue virus vaccine candidates
∆30
rDEN4∆30C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3’5’
rDEN3∆30/31C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3’5’
∆30/31Additional 3’-UTR deletion mutations:
Monovalent dengue vaccine studies in humans
Cohort of 28 healthy adult volunteers (20 vaccinees, 8 placebo recipients)
Vaccine delivered as single subcutaneous dose
Virus titer: generally 103 pfu (101 - 105 for DEN4)
Clinical follow-up
Record temperature t.i.d for 16 days post-vaccination
Seen in clinic every other day for 16 days post-vaccination for physical examination and relevant blood work (CBC with differential, blood chemistries).
Serum for virology collected at every visit through study day 16.
Serum collected for antibody titers at study days 0, 28, 42, and 180.
Immunogenicity following single 103 PFU
dose of a monovalent DEN candidate vaccine
Mean serum neutralizing
antibody titer (range)
Vaccine
No. of
subjects
No.
Infected Day 28 Day 42
% sero-
conversion
DEN1∆30 20 19 444 331 95
DEN2/4∆30 20 20 147 237 100
DEN3-3’D4∆30 20 16 52 72 80
DEN3∆30/31 20 19 136 116 95
DEN4∆30 20 19 139 129 95
D4∆30-200,201 20 19 62 71 95
Vaccine
No. of
subjects
Mean peak
virus titerb
% with indicated clinical signsa
Fever Rashc
Neutro-
peniad
Elevated
ALTe
DEN1∆30 71 1.0 1f 34 42 1 (n=1)
DEN2/4∆30 20 0.6 0 45 35 15 (n=3)
DEN3-3’D4∆30 20 0.6 5f 35 10 5 (n=1)
DEN3∆30/31 20 0.5 0 20 0 5f (n=1)
DEN4∆30 60 0.6 1g 70 23 1 (n=1)
D4∆30-200,201 20 < 0.5 0 20 20 0
Total: 211 1 43 27 3
Placebo 63 - 0 3 6 0
a No volunteer experienced systemic illness defined as > 2 of the following symptoms lasting > 2 days: headache, malaise,
anorexia, myalgia/arthralgia, nausea, eye pain.b Mean peak titer (log10 pfu/ml) calculated only for those volunteers with detectable viremia.c Rash was macular / maculopapular and asymptomatic.d Neutropenia is defined as an ANC of < 1500 /mm3.e ALT < 72 IU/mm3 is defined as normal for males, < 52 IU/mm3 is normal for females. Max. ALT elevation 1.7X ULN.f Unrelated to vaccination.g Max. temperature was 100.5oF on single reading, unconfirmed in clinic.
Monovalent Clinical Summary
(101 - 103 pfu doses)
Goal Findings
Safe
a) For vaccinee Minimal reactogenicity
b) For community Poorly transmissible
c) Genetically stable ∆30 mutation maintained
Immunogenic 80 – 100% seroconversion
Economical Produced at >107 pfu/mL
Administered at 103 pfu
( 1 L = 10 million doses)
Conclusions for the monovalent vaccine candidates
DEN1∆30 DEN2/4∆30 DEN3-3’D∆30 DEN4∆30+ + +
DEN3∆30/31
DEN4∆30-200,201
Formulation TV001 (N = 28, Unblind – Sept 8, 2010):
Current Phase I evaluation of three tetravalent
vaccine formulations
DEN1∆30 DEN2/4∆30 DEN3-3’D∆30+ + +
Formulation TV002 (N = 28, Unblind – Dec 13, 2010):
DEN1∆30 DEN2/4∆30 DEN4∆30+ + +
Formulation TV003 (N = 28, Unblind – Nov 1, 2010):
Clinical summary of DEN tetravalent
vaccine candidates
% with indicated clinical sign
Vaccine
Dose
(log10)
No. of
subjects Fever Rash Headache
Neutro-
penia
Elevated
ALT
TV001 3 20 0 65 45 25 0
Placebo - 8 0 0 50 0 0
TV0021 3 20 0 25 45 30 5
Placebo -
TV003 3 20 0 75 20 20 5
Placebo - 8 0 0 15 5 0
1 TV002 is fully enrolled but has not been unblinded. To date, 20 subjects have been enrolled. The indicated
clinical signs are for the entire cohort, including placebo recipients.
Viremia summary of TV001
Vaccine
components
No. of
subjects
% with
viremia
Mean peak
titera + SE
Mean day of
viremia onset
(range)
Mean
duration
in days
(range)
DEN1∆30
20
40 0.5 ± 0.14 12.1 (9 -16) 2.2 (1 - 5)
DEN2/4∆30 10 0.5 ± 0.17 11.0 (6 - 16) 1.0 (all 1)
rDEN3-3’D4∆30 20 0.6 ± 0.12 11.0 (6 - 14) 2.2 (1 - 4)
rDEN4∆30 40 0.6 ± 0.09 9.6 (5 - 12) 2.0 (1 - 5)
Total % viremicb: 70
a Log 10 PFU/mLb 1 subject viremic with 3 viruses
6 subjects viremic with 2 viruses
Viremia summary of TV003
Vaccine
components
No. of
subjects
% with
viremia
Mean peak
titera + SE
Mean day of
viremia onset
(range)
Mean
duration
in days
(range)
DEN1∆30
20
30 0.7 ± 0.16 9.8 (8 - 12) 2.3 (1 - 5)
DEN2/4∆30 10 0.5 ± 0.00 6.0 (all 6) 1.0 (all 1)
rDEN3∆30/31 40 0.6 ± 0.09 8.2 (5 - 14) 2.6 (1 - 6)
rDEN4∆30 40 0.5 ± 0.00 7.4 (6 - 9) 1.4 (1 – 3)
Total % viremicb: 75
a Log 10 PFU/mLb 1 subject viremic with 3 viruses
3 subjects viremic with 2 viruses
Neutralizing antibody titers: TV-001
DEN1∆30
DEN2/4∆30
DEN3-3’D4∆30
DEN4∆30
Serotype specificity
DEN1 DEN2 DEN3 DEN4
% seroconverted (PRNT > 20) 60 40 45 95
% seroconverted (monovalent) 94 100 80 93
% seroconverted (PRNT > 10) 80 65 60 95
Mean titer (GMT) (PRNT > 10) 54 39 36 154
% with indicated multivalent response (cumulative)
Method Tetra- Tri- Bi- Mono- None
PRNT > 10 30 50 / 80 15 / 95 0 / 95 5
* * *
* Fisher exact test, P < 0.048
Neutralizing antibody titers: TV-002
DEN1∆30
DEN2/4∆30
DEN3-3’D4∆30
DEN4∆30-200,201
Serotype specificity
DEN1 DEN2 DEN3 DEN4
% seroconverted (PRNT > 20) 70 40 40 50
% seroconverted (monovalent) 94 100 80 95
n = 10 n = 10 n = 10 n = 10
% seroconverted (PRNT > 10) 80 40 70 100
Mean titer (GMT) (PRNT > 10) 77 57 22 24
% with indicated multivalent response (cumulative)
Method Tetra- Tri- Bi- Mono- None
PRNT > 10 20 50 / 70 30 / 100 0 / 100 0
* * *
* Fisher exact test, P < 0.045
*
Neutralizing antibody titers: TV-003
DEN1∆30
DEN2/4∆30
DEN3∆30/31
DEN4∆30
Serotype specificity
DEN1 DEN2 DEN3 DEN4
% seroconverted (PRNT > 20) 100 45 60 100
% seroconverted (monovalent) 94 100 95 93
% seroconverted (PRNT > 10) 100 50 85 100
Mean titer (GMT) (PRNT > 10) 62 44 36 65
% with indicated multivalent response (cumulative)
Method Tetra- Tri- Bi- Mono- None
PRNT > 10 40 50 / 90 10 / 100 0 / 100 0
* *
* Fisher exact test, P < 0.01
Neutralizing antibody titers: TV-003
DEN1∆30
DEN2/4∆30
DEN3∆30/31
DEN4∆30
Serotype specificity
DEN1 DEN2 DEN3 DEN4
% seroconverted (PRNT > 20) 100 45 60 100
% seroconverted (monovalent) 94 100 95 93
% seroconverted (PRNT > 10) 100 50 85 100
% seroconverted (ChimeriVax) 36 13 45 42
% seroconverted (GSK)* 17 65 30 65
Mean titer (GMT) (PRNT > 10) 62 44 36 65
% with indicated multivalent response (cumulative)
Method Tetra- Tri- Bi- Mono- None
PRNT > 10 40 50 / 90 10 / 100 0 / 100 0
* *
* Sun, et al., 2009, Human Vaccines 5:33-40.
Some individual neutralizing antibody titers
of interest
Mean peak PRNT (GMT)
Subject no. DEN1 DEN2 DEN3 DEN4
268.01.01 88 523 182 430
Some individual neutralizing antibody titers
of interest
Mean peak PRNT (GMT)
Subject no. DEN1 DEN2 DEN3 DEN4
268.01.01 88 523 182 430
268.01.05 < 5 < 5 < 5 < 5
Some individual neutralizing antibody titers
of interest
Mean peak PRNT (GMT)
Subject no. DEN1 DEN2 DEN3 DEN4
268.01.01 88 523 182 430
268.01.05 < 5 < 5 < 5 < 5
268.01.14 6 183 28 276
Some individual neutralizing antibody titers
of interest
Mean peak PRNT (GMT)
Subject no. DEN1 DEN2 DEN3 DEN4
268.01.01 88 523 182 430
268.01.05 < 5 < 5 < 5 < 5
268.01.14 6 183 28 276
268.01.21 87 < 5 71 103
Some individual neutralizing antibody titers
of interest
Mean peak PRNT (GMT)
Subject no. DEN1 DEN2 DEN3 DEN4
268.01.01 88 523 182 430
268.01.05 < 5 < 5 < 5 < 5
268.01.14 6 183 28 276
268.01.21 87 < 5 71 103
268.01.27 151 30 < 5 344
Some individual neutralizing antibody titers
of interest
Mean peak PRNT (GMT)
Subject no. DEN1 DEN2 DEN3 DEN4
268.01.01 88 523 182 430
268.01.05 < 5 < 5 < 5 < 5
268.01.14 6 183 28 276
268.01.21 87 < 5 71 103
268.01.27 151 30 < 5 344
268.01.12 < 5 < 5 39 422
Some individual neutralizing antibody titers
of interest
Mean peak PRNT (GMT)
Subject no. DEN1 DEN2 DEN3 DEN4
268.01.01 88 523 182 430
268.01.05 < 5 < 5 < 5 < 5
268.01.14 6 183 28 276
268.01.21 87 < 5 71 103
268.01.27 151 30 < 5 344
268.01.12 < 5 < 5 39 422
268.01.15 48 < 5 < 5 74
Some individual neutralizing antibody titers
of interest
Mean peak PRNT (GMT)
Subject no. DEN1 DEN2 DEN3 DEN4
268.01.01 88 523 182 430
268.01.05 < 5 < 5 < 5 < 5
268.01.14 6 183 28 276
268.01.21 87 < 5 71 103
268.01.27 151 30 < 5 344
268.01.12 < 5 < 5 39 422
268.01.15 48 < 5 < 5 74
268.01.13 11 7 10 352
Is this individual primed for ADE disease?
• Unknown if 1:7 is protective (or would restrict replication)
• Boost would likely occur within 6 months
• Severe disease has not been reported by others during vaccine follow-up in endemic
areas
Conclusions from tetravalent study
1. The tetravalent mixtures are safe.
2. Viremia remained very low.
3. 70 - 90% of subjects had at least a trivalent antibody response.
4. It appears that there is very little cross reactivity between
strains.
5. Booster immunization?
May be needed to achieve a tetravalent response
Will likely increase cross reactivity
Should increase antibody durability
1. Complete initial evaluation of the tetravalent vaccine mixtures,
with second vaccination at 6 months.
2. Provide each of the four components to our licensees in Brazil,
India, and Vietnam.
3. Determine the number and the timing of doses needed for an
optimal antibody response.
4. Evaluate tetravalent vaccine in endemic region.
Safety
Age de-escalation
Immediate clinical plans for the development
of the tetravalent dengue virus vaccine