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

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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

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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)

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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

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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

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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

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>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)

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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)

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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

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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)

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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