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Development of a VP6 subunit rotavirus vaccineA dual role of VP6 as a vaccine antigen and an adjuvant
Dr. Vesna BlazevicHead of Laboratory
Vaccine Research Center
University of Tampere
1
30 August 2018, Minsk13TH INTERNATIONAL ROTAVIRUS SYMPOSIUM
Rotavirus (RV) VP6
http://www.reoviridae.org/dsRNA_virus_proteins/Rota%20cut-away.gif
VP6RV Triple-layered RV particle:
1. Inner capsid VP2
2. Intermediate capsid VP6
• Highly conserved protein• Groups (A-H)• Subgroups (SGI, II, I+II, non I/II)
3. Outer capsid VP7 with VP4 spikes
-> a dual role of VP6 as a subunit rotavirus vaccine and an adjuvant in a combination norovirus (NoV) + RV vaccine candidate developed by our laboratory
2
3
Rationales for a non-live subunit RV vaccine
• No adverse issues of live RV vaccines• No competition of uptake with enteric viruses in the gut • Live RV vaccines (RV1 and RV5) show lower efficacy in Africa and Asia• Live RV vaccines show decline of efficacy in the 2nd year in developing
countries• Genetic background of the population not critical (e.g. secretors/non-
secretors)
4
Combination NoV+RV vaccine: recombinant NoV virus-like particles (VLPs) + RV VP6
→ Non-live subunit vaccine against childhood gastroenteritis
→ All vaccine antigens are oligomeric proteins produced in insect cells by recombinant baculovirusexpression vector system (BEVS)
NoV VLPs~30-40 nm
VP6 nanotubes (VP6T)~75 nm, 0.2-1 µm long (pH 7)
or
VP6 nanospheres (VP6S)~75 nm (pH 4)
100 nm
VP1
http://www.defendingfoodsafety.com
capsid protein VP1
NoV
http://www.reoviridae.org/dsRNA_virus_proteins/Rota%20cut-away.gif
VP6
capsid protein VP6
RV
BEVS
100 nm
or
capsid VP2+
VP2
Double-layered 2/6-VLPs~65 nm
~100 nm
→ M. Estes et al., J Virology 1987 (rVP6 produced in BV expression system)
RV rVP6
Trivalent combination vaccine formulations -extensively studied in our laboratory
5
NoV GII.4 VLPs + GI.3 VLPs + RV VP6 oligomers (VP6T or VP6S or dlVLPs)
a representative of GI and GII NoVs
no external adjuvants!
GII.4 VLPs
GI.3 VLPs
GII.4 VLPs
GI.3 VLPs
VP6 T dlVLPs
The combination vaccine induces strong NoV and RV type-specific and cross-reactive immunity
6
PLOS One. 2013 Jul 26;8(7):e70409.
Strong cross-reactive serum antibodies to heterologous RV strains
Low levels of IgA in feces of IM immunized mice• Rapid increase after live RV challenge
0.0
0.5
1.0
1.5
2.0
2.5 Trivalent (GII-4+GI-3+VP6)
0.0
0.2
0.4
0.6
0.8
1.0VP6 IMVP6 INControl IMControl IN
0.0
0.1
0.2
0.3
0.4VP6 IMVP6 INControl IMControl IN
VP6-specific mucosal IgG VP6-specific mucosal IgAVP6-specific serum IgG
OD 4
90nm
OD 4
90nm
Sample dilution Sample dilution
RV antigen shedding
0
0.1
0.2
0.3
0.4
0.5
0.6
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8
OD
450n
m
VP6 IN
VP6 IM
Control
65 % reduction
66 % reduction
Days post-challenge
7
>50 % reduction in RV antigen shedding in the stool considered significant protection from virus challenge
Protection correlated with VP6-specific serum IgA levels
VP6 protects mice from live RVEDIM challenge
No traditional neutralizing antibodies, instead non-serotype specific protective immunity
• VP6-specific cytotoxic T lymphocytes (CD8+ T cells) (H Greenberg’s group)• VP6-specific CD4+ T cells (R Ward’s group)
• Direct cytotoxic mechanism and anti-viral cytokines
• polymeric/secretory IgA antibodies (H Greenberg, J Crowe)• Intracellular neutralization by VP6-specific IgA antibodies following
transcytosis
Mechanisms of VP6 protection
VP6-specific pIgA inhibits RV replication at the transcription level by blocking channels on dlRVparticles and preventing RV mRNA release (Aiyegbo et al. 2013, 2014)
8
50
• RV-specific serum IgA antibody levels were shown to correlate to RV1 and RV5 vaccines efficacy (Patel et al. 2013)
Correlates of protection in children
Early live bovine RV vaccine RIT4237 induced the strongest response against VP6 (Svensson et al. 1987)
10
100
1000
10000
100000
10 100 1000 10000 100000
Seru
m Ig
A ag
ains
t rVP
6
Serum IgA against Wa
r=0.683(p
VP6 adjuvant effect on NoV immunity
0.3 µg NoV VLP + VP6+ co-delivery (mix)
Study design
NoV GII.4 -specific T cell responsesNoV GII.4-specific serum IgG
IM 0 3 5
T
10
0.0
0.5
1.0
1.5
2.0
2.5
3.0
1:10
0
1:20
0
1:40
0
1:80
0
1:16
00
1:32
00
1:64
00
1:12
800
1:25
600
1:51
200
1:10
2400
1:20
4800
1:40
9600
1:81
9200
OD
490n
m
Serum Dilution
0.3µg GII-4
10µg GII-4
0.3µg GII-4 + 10µg VP6
0
50
100
150
200
0.3µg VLP 0.3µg VLP +10µg VP6T
0.3µg VLP +10µg VP6S
Ctrl
IFN
-γSF
C /
106
sple
nocy
tes
Experimental Group
GII.4 peptide poolOVA peptide
VLP
VLP VP6T/S
(Blazevic et al. 2015, Malm et al. 2017)
RV VP6 acts as a delivery vehicle for NoVVLPs
0.0
0.5
1.0
1.5
2.0
2.5
3.0
OD
490
nm
Serum dilution
NoV GII.4 -specific IgG titers0.3 µg GII.4 VLP 10 µg VP6 T
+
contralateralsites
co-delivery
1h betweeninjections
IM 0 3 5
T
11
(Malm et al. 2017)
VP6 is internalized by antigen presenting cells (APC)
→ Intracellular staining with anti-VP6-FITC ab shows that VP6 is internalized by macrophages and dendritic cells (DC)
12
Anti-VP6 FITC
RAW 264.7
RV VP6
CM
Anti-VP6-FITC
iBMDC
RV VP6 activates macrophages and DC
Mean fluorescence intensity (MFI)
CM BSA VP6VP6 %
increase
MHC II 169 151 376 122
CD80 126 118 302 140
→ increased cell surface expression of antigen presentation and co-stimulatory molecules
CM VP6
13
0
5000
10000
15000
TNF-
a (p
g/m
l)
TNF-α production by RAW 264.7 cells
CM
BSA 50 ug/ml
VP6 100 ug/ml
0
1000
2000
3000
24h
IL-6
(pg/
ml)
IL-6 production by JAWSII cells
CM
BSA 50 ug/ml
VP6 100 ug/ml
→ Induced cytokine production by macrophages and DC
(Malm et al. 2016)
14
RV VP6-specific immune response protects from heterologous RV challenge in mice
Importance of non-serotype-specific immunity induced by VP6 in protection from RV infection
RV VP6 acts as an adjuvant for norovirus VLPs
RV VP6 activates antigen presenting cells (APC)
RV VP6 is an excellent subunit vaccine candidate against RV
Conclusions VP6 VP6 T VP6 S
Acknowledgements
• Vaccine Research Center• University of Tampere, Finland
• Suvi Heinimäki• Maria Malm• Kirsi Tamminen• Eeva Jokela• Sanna Kavén• Nina Koivisto• Timo Vesikari
• Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, México
• Ana Ruth Pastor• Laura A. Palomares
• Facultad de Medicina, Universidad Autónoma del Estado de Morelos, Morelos, México
• Vanessa López-Guerrero• Fernando Esquivel-Guadarrama
Thank you15
http://www.umnpharma.com/en/index.htmlhttp://www.umnpharma.com/en/index.html
Development of a VP6 subunit rotavirus vaccine�A dual role of VP6 as a vaccine antigen and an adjuvant Slide Number 2Rationales for a non-live subunit RV vaccineSlide Number 4Trivalent combination vaccine formulations -extensively studied in our laboratoryThe combination vaccine induces strong NoV and RV type-specific and cross-reactive immunitySlide Number 7Mechanisms of VP6 protectionSlide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Conclusions Slide Number 15