2. Outline
1- Dengue virus, dengue vaccination, and project
concept.
2-Broad neutralizing responses elicited by flavivirus
infections.
3- Generation of a complementary anti-dengue
immunization strategy.
4- Conclusion and future directions.
2
3. Dengue:a continuingglobal threat
Dengue is endemic in at least 100 country; 40% of
the world’s population, live in areas where there is a
risk of dengue transmission. Causing about 100
million infection yearly and 22,000 death, mostly
among children.
Simmons CP et al, 2012
3
4. DengueDisease
Warning signs
Persistent vomiting
Fluid accumulation
Bleeding
Asymptomatic
OR
Dengue fever
High fever
Muscle pain
Bone pain
Rash
Nausea
vomiting
Dengue hemorrhagic fever (DHF)
and Dengue sock syndrome (DSS)
Internal bleeding
Low platelet count
Death within 24 to 48 hour
Severe Dengue
80%
20%
5%
4
5. DengueVirus
• A member of Flaviviridae family
• An enveloped, positive polarity, single stranded RNA virus
• Infects human through Aedes aegypti mosquito bite
• There are four serotypes of Dengue virus ( DV1-4)
• The most prevalent arthropod-borne pathogen
http://www.abc.net.au
5
6. Denguevaccinesat a crossroad
Seven decades of dengue vaccine research have shown how challenging it is to
develop a highly efficacious vaccine that protects against all four serotypes.
Vaccine candidate Phase I Phase II Phase III
DNA vaccine
Recombinant
subunit
Purified
inactivated
Live
attenuated
Chimeric live
attenuated
Adapted from nature review| microbiology
6
7. The benefitsand the risks
of dengue first approved
vaccine
(Dengvaxia®bySanofiPasteur)
• Live attenuated tetravalent chimeric vaccine.
• Made by replacing PrM and E genes of YFV 17D strain
vaccine with those from the four DV serotypes.
• Approved in Mexico, the Philippines, Brazil, El Salvador and
Indonesia.
• Two parallel phase III clinical trails were done in Asia (CYD14)
and in Latin America (CYD15). Those clinical trial results
showed:
Uneven protection
Low efficacy in seronegative
Enhancement of the disease in young children
7
8. Unevenprotection
0
10
20
30
40
50
60
70
80
90
DV 1 DV 2 DV 3 DV 4 DV 1-4
CYD 14
CYD 15
Pool
VaccineEfficacy%
Adapted from S. Thomas Dengue vaccine (CYD-TDV) clinical trials result. Meeting of the strategic Advisory Group of Experts (SEGA), 14th April 2016
8
10. Enhancementof dengue infectionamong
children 2 to 9 YO
0
20
40
60
80
2-5 yrs 6-11 yrs 12-16 yrs
VaccineEfficacy%
0
10
20
30
40
50
60
70
2-5 yrs 6-8 yrs 9-11 yrs 12-16 yrs
0
5
10
15
20
25
2-5 yrs 6-8 yrs 9-11 yrs 12-16 yrs
Numberofcases
Numberofcases
CYD 14
Control
Hospitalized Dengue per age groups Severe Disease per age groups
Adapted from S. Thomas Dengue vaccine (CYD-TDV) clinical trials result. Meeting of the strategic Advisory Group of Experts (SEGA), 14th April 2016
10
11. Dengvaxia®acted like a natural silent
infection
WHO| reports. Comparative modeling of dengue vaccine public health impacts (CMDVI) 17th March 2016
11
12. Achievinga safe and effectiveDenguevaccine
in youngchildren
Using the current MV immunization scheme to prime anti-
flaviviral neutralizing immunity in infants may be a highly
convenient and cost-effective strategy to robustly enhance the
immunogenicity and efficiency of subsequent tetravalent
Dengue vaccine approaches.
Naïve
Seronega ve
infant
MV-DV vaccine
cocktail
Seropos vity
Tetravalent DV
Vaccine
12 month
old
18 month
old
five months
Primary infec on
No sever Dengue
Long life protec on
Primary infection
No severe Dengue
12
15. Recently, the cross-reactivity in immune
responses to flavivirus infection raised an
interesting question in terms of vaccine
development: does cross-immunity provide
cross-protection within the same family?
Wanwisa D. et al. 2016
Jieqiong, L. et al. 2016
Takasaki, T. et al. 2003
Mangiafico, J. et al. 2002
Tesh, R. et al. 2002
Price, W. H. & Thind, I. S. 1971
Sather, G. E. & Hammon, W. M. 1970
Tarr, G. C. & Hammon, H. W. 1974
15
17. Is it possibleto see crossneutralizationwith
our animal model “HuCD46Ge-IFNarKO mice”
Day 0: intra-peritoneal
inoculation of 105 PFU/ml virus
Day 32: Euthanize
HuCD46Ge-IFNarKO mice Immunization
Schedule
DV1 OR DV2 OR DV4 OR YFV
express a human receptor for vaccine strains of MV
with human-like tissue specificity
a type I interferon-deficient background permissive of
viral replication.
17
18. YY
Y
Heat inactivatedserum
Log neutralization index(LNI) Plaque reduction neutralization Titer50 (PRNT50)
YY
Y
Y
Y
Y
Neutralization
Incubated 1 hour at 37°C
Titration of remaining infectivity as it
compared to non-immune serum
Virus of known
titer
Non-diluted Serum
of immunized animal
25ul 25ul
YY
Y
YY
Two fold dilutions
Serum of
immunized animal
50 PFUs
of virus
1:20 1:1280
Y
Y
Y
Y
Y
Y
Y
1:10 1:2560
2 fold serial dilutions
Neutralization
Incubated 1 hour
at 37°C
Plaque assay to determine the dilution
has 50% reduction in plaque
Quantifyingneutralizingantibodies
LogNeutralizationIndex(LNI)VSplaquereductionneutralizationtiter(PRNT50)
Experimental design
18
27. ExpressionofDengue2 glycoproteinsbyrecombinantviruses
Anti-MV-Rhodamine Anti-DV2-FITC MERGE
MVvac2 DV2 MOCK
Anti-E
Anti-PrM
Anti-E
Anti-PrM
-D2(prME)N-D2(prMEsol)NControls
MV MOI of 0.06
DV MOI of 1
48 hrs Time post-infection
Primary Ab
Mab Anti-DV-E-Mouse
1:50
Mab Anti-DV-prM-Mouse
1:50
Pab Anti-MV-N-Rabbit
1:300
Overnight at 4 C
Secondary Ab
Anti-mouse-FITC
Anti-Rabbit-Rhodamine
1 hour at room temp.
27
28. ExpressionofDengue2 glycoproteinsby
recombinantviruses
Vero/hSLAM cells
Experimental Design
at 80% cytopathic effect in
infected cells, collect cell lysates
SDS-PAGE & protein
immunoblot
+ -
MVvac2
MV-DV2(prME)N
MV-DV2(prMEsol)N
Mock
50
75
50
kDa
75
Anti MV N
Anti Actin
Anti Den E
-D
2(prM
E)N
-D
2(prM
Esol)N
M
V
vac2
M
ock
A 4 kDa difference in the
electrophoretic migration of the
vectored DV2 Esol molecular weight
certainly obeys to the 45 amino acid
carboxyl terminus truncation of E as
expressed by D2(prMEsol)N.
1:100
1:5000
1:10000
28
29. Analysisof vectoredDengue2E glycoprotein
glycosylation
Glycosidase mode of act
Endoplasmic Reticulum Trans-Golgi
High Mannose
✔ Endo H
✔ PGNASF
Hybrid
✖ Endo H
✔ PGNASF
Complex
✖ Endo H
✔ PGNASF
75
100
50
kDa
Anti MV H
Anti Den E
Endo H
PNGase F+
+ -
-+
+ -
- -
- -
-
-D2(prME)N -D2(prMEsol)N
*
* *
EndoH-resistant DV2E is observed
in D2(prMEsol)N but not in
D2(prME)N infected-cells lysates,
which is consistent with a protein
in route to secretion.
29
44. By combining DV2
and DV4 insertions we were able to enhance the homologous and heterologous
neutralizing immunity. This effect is not due to transient cross reactivity since
single component immunization did not reach the observed neutralizing titers.
Broad neutralizing anti-Falvivirus
Anti-measles neutralizing antibodies
Major finding
44
45. Does serum form animal
inoculated with MV-DV2/4
cocktail enhance DV1 infection
of FC receptor bearing cells?
45
46. SerumfromanimalinoculatedwithcocktailofrecombinantMV-
DV2/4dosenotenhancetheinfectionofmousemonocytewithDV1
-3H5 clone was extracted from culture
of mouse B cells hybridoma (HB-46).
- Mab MCA2277 , 1.0 mg/ml 3H5 clone
1
10
100
1000
Full dose Half dose Clone
3H5
Mab
MCA2277
Unrelated
serum
1:100
1:10000
Dengue1virustiterlog10(PFU/ml)
N.D N.D
3
2
1
0 N.D
Experimental design
DV1
Heterotypic
antibodies
1 hr-37C
Heterotypic antibody binds
virus but not neutralizing
Fc receptor
Mouse monocyte
DV replicates poorly in Fc receptor bearing
cells in absence of cross reactive antibodies
Enhancement of infection
Plaque assay
to determine
viral load
46
N.D
Not. Detected
47. Future Directions
• Priming flaviviral immunity at an early age may prove
convenient and complementary to many other flavivirus
vaccine approaches.
• Cocktail of recombinant measles expressing major
glycoproteins of phylogenetically related flavivirus could be a
effective in protection against multiple flavivirus infections.
47
48. Acknowledgments
Funding
Arizona Board of Regents
School of Life Sciences
Committee
Jorge Reyes del Valle
Hugh Mason
Douglas Lake
Valerie Stout
Wayne Frasch
Reyes del Valle Laboratory
Ivonne Ceballos Olvera
Emily Julik
Indira Harahap
Reagents
Roberto Cattaneo
48
