Clinical evaluation of the LID live attenuated …€™ C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3’ ∆30 rDEN3-3’D4∆30 3’-UTR swap with rDEN4∆30: DEN4 5’ C prM E NS1 NS2A

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.


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?


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.


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







antibody with broad


serotypes.

homotypic

homotypic


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







antibody with broad


serotypes.

homotypic

homotypic

heterotypic

heterotypic


But wait!

Dengue immunity


infection is not without

its problems.

Primary

infection

Secondary

infection

(different serotype)

Antibody

dependent

enhanced

disease

?


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


∆30rDEN1∆30

rDEN4∆30


∆30 rDEN1/4∆30

rDEN2/4∆30

rDEN3/4∆30


30 deletion mutation in 3’-UTR:

Antigenic chimeras (rDEN4∆30 background):

prM and E region from wildtype DEN1, DEN2, or DEN3


∆30rDEN1∆30

rDEN4∆30


∆30 rDEN1/4∆30

rDEN2/4∆30

rDEN3/4∆30


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



∆30

rDEN3-3’D4∆30

3’-UTR swap with rDEN4∆30:

DEN4

C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS55’ rDEN3


∆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


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


DEN1∆30

DEN2/4∆30

DEN3-3’D4∆30

DEN4∆30-200,201


DEN1 DEN2 DEN3 DEN4



n = 10 n = 10 n = 10 n = 10





PRNT > 10 20 50 / 70 30 / 100 0 / 100 0

* * *


*


DEN1∆30

DEN2/4∆30

DEN3∆30/31

DEN4∆30


DEN1 DEN2 DEN3 DEN4







PRNT > 10 40 50 / 90 10 / 100 0 / 100 0

* *



DEN1∆30

DEN2/4∆30

DEN3∆30/31

DEN4∆30


DEN1 DEN2 DEN3 DEN4




% seroconverted (ChimeriVax) 36 13 45 42

% seroconverted (GSK)* 17 65 30 65




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


of interest



268.01.01 88 523 182 430

268.01.05 < 5 < 5 < 5 < 5


of interest



268.01.01 88 523 182 430

268.01.05 < 5 < 5 < 5 < 5

268.01.14 6 183 28 276


of interest



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


of interest



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


of interest



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


of interest



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


of interest



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

Documents

Clinical evaluation of the LID live attenuated …€™ C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 3’ ∆30 rDEN3-3’D4∆30 3’-UTR swap with rDEN4∆30: DEN4 5’ C prM E NS1 NS2A