DENGUE VACCINESWHERE ARE WE NOW?

SCOTT B HALSTEAD, MDSCOTT B HALSTEAD, MD

DENGUE VACCINE INITIATIVEINTERNATIONAL VACCINE INSTITUTE

SEOUL,KOREA

STATUS

• Vaccine ownership change:

CDC Inviragen Takeda PharmaceuticalsTakeda Pharmaceuticals

Current candidate tetravalent dengue vaccines in human trials

• LIVE ATTENUATED– Sanofi – dengue 1 – 4/yellow fever chimera– Takeda – mutant dengue 2, dengue 1,3, 4/dengue – Takeda – mutant dengue 2, dengue 1,3, 4/dengue

2 chimera– NIH – mutagenized dengue 1, 3, 4, dengue 2/4

chimera• INACTIVATED

– GSK – inactivated whole virus– Merck – subunit vaccine

STATUS

• Sanofi – in phase III• Takeda – in phase II, nearing phase III• NIH – in prolonged phase I, Butantan

vaccine nearing phase I and II.vaccine nearing phase I and II.• GSK – in phase I• Merck – in phase I

STATUS

• Sanofi phase IIb clinical trial results.

– Sabchareon et al Lancet Sept 2012

Results of CYD23 phase IIb clinical trial

• Three doses of vaccine in children more than 55 - 60% of whom had DENV neutralizing antibodies at baseline, raised multitypic antibodies in 95 – 100% of children 3 weeks after 3 rd dose.

• These antibodies did not protect against DENV-2.

• Nor did protection against DENV 1, 3 or 4 increase with successive doses.

ETIOL VACC AT RISK

CONTR AT RISK

VACCCASES

CONTROLCASES

EFFICACY

DENV 1 5343 2666 14 18 61.2

DENV 2 5312 2662 52 27 0.9

DENV 3 5348 2667 4 11 81.9

CYD 23 efficacy – intention to treat

DENV 3 5348 2667 4 11 81.9

DENV 4 5353 2679 1 5 90.0

SABCHAROEN et al LANCET Nov 12

CYD 23 efficacy after 1, 2 or 3 doses or any dose.

ETIOLOGY AFTER 1 DOSE

AFTER 2 DOSES

AFTER 3 DOSES

ANY

DENV 1 0 87.5 55.6 61.2

DENV 2 25.1 0 9.2 0.9

DENV 3 80.1 87.5 75.3 81.9

DENV 4 100 na 100 90.0

What went wrong?

• Need to understand the nature of • Need to understand the nature of protective immunity in human dengue virus infections.

Three types of protective DENV immunity:

• Homotypic – protection on challenge with same virus.

• Heterotypic – protection following one infection for a different dengue virus.infection for a different dengue virus.

• Multitypic – protection following infection with two or more dengue viruses.

Homotypic flavivirus immunity recently demonstrated to be mediated by attachment of antibody to intact virion. antibody to intact virion.

DENV 1 – 3 escapemutants identifyinglocation of Nt antibodyattachment tovirions.De Alwis et alPNAS 2012

QUATERNARY STRUCTURE OF DENV Nt Abs

PNAS 2012

Fab West Nile Ntantibodies attachedto WN virions.Kaufmann et alPNAS 2010

Fab 14C10

7 angstrom resolution map of Fab 14C10 to DENV-1 vi rion

E proteinlayer

Tm helices

membrane –outer leaflet

membrane – inner leaflet

Courtesy of Sheemei Lok, Duke-NUS

But, T cells may contribute to homotypic protective

immunity

• Mice immunized with DENV-2 NS3 DNA were able to resist lethal DENV -2 i.c. challenge.to resist lethal DENV -2 i.c. challenge.

• Costa et al PLoS ONE 6: e 25685, 2011

• Mice immunized with NS 2A, 4B 5 CD 8+ T cell epitopes resisted DENV-2 challenge.

• Yauch et al J Immunol 2009

IN CYD 23 THERE WAS NO MONOTYPIC PROTECTION AGAINST DENGUE 2.

WHY NOT?

1. Interference of “takes” when multiple vaccine viruses are given at a single s.c. vaccine viruses are given at a single s.c. site?

2. Vaccine failure?3. Absence of heterotypic/multitypic

protection?

1) INTERFERENCE WHEN MULTIPLE STRAINS ARE INOCULATED AT STRAINS ARE INOCULATED AT

ONE SITE

Interference occurs when vaccine given at one or multiple sites

Guy et al AJTMH 2009

2. Vaccine failure. Was monotypic dengue 2 chimera a truly protective

vaccine?

After one dose YF/D2 in rhesus, 56 days later challenge with wt DENV 2 –

no viremia

Guirakhoo et al J. Virol 2000

Wild -type DENV 2challenge day 63.ManyanamnesticNt antibodyresponses. responses.

Guirakhoo et alJ Virol 2000

Based on anamnestic antibody responses, susceptible monkeys

vaccinated with CYD -2 failed to be “solidly protected” * to dengue 2

challenge.

* For protection criteria see: Halstead et al AJTMH 22: 365-74,1973

3. Absence of heterotypic/multitypic

protection protection

Evidence of heterotypic immunity in humans

• Short interval DENV -1protection against DENV -2 infection. Sabin 1950

• For 9 months DENV 1 immunity modulates DENV 2 disease. Sabin, 1950modulates DENV 2 disease. Sabin, 1950

• Of all second dengue infections, only 2 - 4% are hospitalized, 8 – 10% are mild/DF; the rest are inapparent (~85 %) Multiple reports .

Evidence of heterotypic immunity in humans

• Mild disease when second infections occur at short intervals; severe disease occurs when second dengue infections occur at longer intervals. Anderson et occur at longer intervals. Anderson et al JID 2013; Reich et al J Roy Soc Interf 2013; Gibbons et al AJTMH 2007; Guzman et al PAHO J Epidemiol 2002

How to measure heterotypic protective immunity?

• Use primary human mononuclear phagocytic (myeloid) cells.

BANGKOK STUDYKliks et al AJTMH 40:444, 1989.

• 40 Bangkok school children had documented secondary DEN 2 infections (pre -infection blood sample contained dengue antibodies).contained dengue antibodies).

• 7 were hospitalized; 33 silent.• Undiluted pre -infection sera tested for

neutralization or enhancement in primary human monocyte cultures.

ADE BLOCKED BY SOME HETEROTYPIC ANTIBODIES

40 school children with secondary dengue infections, Bangkok, 1980

Undiluted sera Hospitalized Silent Undiluted sera Hospitalized Silent DEN 2 PRNT + 1 29 DEN 2 PRNT - 6* 4*

7 33

* Undiluted sera enhanced growth of DEN 2 in human monocyte cultures. Kliks et al AJTMH 40:444-451, 1989

HETEROTYPIC IMMUNITY SUMMARY

• Heterotypic protective immunity is very strong immediately after DENV infection [DENV 1 protects against DENV 2].DENV 2].

• Heterotypic immunity reduces severity of mild or severe dengue disease over many years, gradually waning with time.

HETEROTYPIC IMMUNITY SUMMARY

• Heterotypic immunity may be reflected in the fact that only 2 – 4% of secondary dengue infections proceed to DHF.to DHF.

• With low levels of heterotypic immunity overt disease attack rates may be 100% during secondary infections.

HETEROTYPIC IMMUNITY SUMMARY

• Heterotypic protective immunity may be partially mediated by cross -neutralizing antibodies that are best measured in FcR -bearing cells. measured in FcR -bearing cells.

• Heterotypic DENV antibody titers continually decline over long periods of time.

• Heterotypic immunity may be partially mediated by T cells/innate immunity.

Multitypic Protection• Second infections raise protective multitypic

immunity - third dengue infections rarely develop DHF. Gibbons R et al AJTMH 77: 910-3. 2007

• In mice, second dengue infections produce protective highly avid, cross -reactive protective highly avid, cross -reactive antibodies. Zompi S et al J Immunol 188:404-16,2012

• In humans, second dengue infections produce protective, highly avid, cross-reactive antibodies. Tsai WY et al J Virol 2013 (in press)

DHF BANGKOK CHILDRENS HOSPITAL

500

600

700

0

100

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300

400

<1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16AGE (YEARS)

1973-791980-891990-99

DSS in a 6 month-old infant with primary dengue infection. Vietnam

Infant DHF/DSS - 5% of all cases

CHILDREN'S HOSPITAL, BANGKOK, THAILAND

25

30

35

40

1987

1990

0

5

10

15

20

0-2 2-4 4-6 6-8 8-10 10-12AGE (months)

1997

1998

Infants are protected for a couple of months early in life by passively transferred dengue antibodies. An example of the neutralizing

antibody titers observed in cord blood sera is given on the next slide. given on the next slide.

D-1 D-2 D-3 D-4 JE

A 640 160 160 320 160

B 1280 640 1280 1280 2560

CORD BLOOD DENGUE NEUTRALIZING ANTIBODIES

B 1280 640 1280 1280 2560

Why no CDY 23 heterotypic/multitypic

protection?• Perhaps neutralizing antibodies from

vaccinees should have been measured in primary human in primary human monocytes/macrophages?

WHY NO CYD 23 HETEROTYPIC/MULTITYPIC

PROTECTION?

• Weak dengue -specific T cell immunity? • Weak dengue -specific T cell immunity? CYD viruses express only yellow fever non -structural antigens.

Summary:Dengue Protective Immunity

• Monotypic immunity most likely due to antibodies with T cells in secondary role.

• Heterotypic immunity, significant role • Heterotypic immunity, significant role for T cells with secondary role for antibodies.

• Multitypic immunity, role for T cells and antibodies.

Problems to be faced/solved

• Can effective and lasting protection be mediated solely by DENV structural antigens?

• Or does protection require exposure to • Or does protection require exposure to DENV non -structural antigens?

• Is protective T cell immunity DENV type -specific?

Implications

• Sanofi vaccine presents structural, no dengue NS peptides.

• Takeda vaccine presents structural but only DENV -2 NS peptides.

• NIH vaccine presents structural and DENV-1, -3 and -4 NS peptides.

• GSK vaccine presents mostly DENV structural peptides.

• Merck vaccine presents structural, no NS DENV peptides.

Patience!