FMDV immunology to improve

vaccines and vaccination

Early induction of neutralising antibodies- suggesting a T-cell independent antibody response

Depletion of CD4+ T cells

during acute infection with FMDV

VT74-CC30/CC51

VT77-CC30/CC51

VT76-CC30/CC51

VT75-CC30/CC51

CD21 CD21

CD21 CD21

CD4 CD4

CD4CD4

FMDV capsid: T independent and T dependent epitopes

Outcome of this experiment

IgG titre (Day 16pb)

Plasma

cell

number

(Day 3pb)

IgG titre (Day 16pb)

Memory

cell

number

(Day 5pb)y = 0.0002x + 1194.1

R2 = 0.08490

500

1000

1500

2000

0 500000 1000000 1500000

y = -0.0084x + 16423

R2 = 0.379

0

5000

10000

15000

20000

0 500000 1000000 1500000

No correlation between the Ab titres and the peak number of plasma cells or memory

B cells generated.

BoostOva Day 6/7Day 3/4Day 20

Day 5/6

Determination of the kinetics of plasma cells, memory B cells and Ab titre in the

blood following immunisation (and boost immunisation) with ovalbumin:

Plasma cells

Memory B cells

Ab titre

Primary response Secondary response

Calf # 703064 (Immunised with TNP-CGG in QuilA)

0

20,000

40,000

60,000

80,000

100,000

120,000

0 20 40 60 80 100 120

Days

An

ti-T

NP

-ova t

itre

sIgG

IgM

Calf # 503069 (Immunised with TNP-FICOLL in QuilA)

0

1,000

2,000

3,000

4,000

5,000

0 20 40 60 80 100 120

Days

An

ti-T

NP

-ova t

itre

s

IgG

IgM

Immunisation Boost

(day 58)

Immunisation Boost

(day 58) Plasma cells

(day 4pb)

Calf # 703064 (immunised with TNP-CGG)

0

2,000

4,000

6,000

8,000

AS

C n

um

be

r

Calf # 503069 (immunised with TNP-FICOLL)

0

1,000

2,000

3,000

AS

C n

um

be

r

T-dependent vs T-independent

responses

TD

Response

TI

Response

Memory

B cells

(day 6pb)

Immune response to vaccination- rapid induction of antibody

- variable/ short duration of immunity

- variable CD4 T cell response

The way forward

Improve understanding of immune response

Target antigen to antigen presenting cells

Stabilise vaccine antigen

0

50

100

150

200

250

300

Medium O1-BFS O1-BFS + immune

IgG

O1-BFS UV O1-BFS UV +

immune IgG

Co

un

ts p

er

min

ute

x 1

03

*

Dendritic cell targeting of FMDV antigen can be improved

University of

Oxford

Ray Owens

E. coli

mammalian cells

IAH-Epidemiology

Javier Castillo-Olivares/

Satya Parida

MVA

Adenovirus

Sendai virus

University of

Reading

Xiaodong Xu/

Ian Jones

Dr. Saravannan

IVRI India

baculovirus

John Innes Centre

George Lomonossoff

plant virus

yeast

IAH-Microbiology

Alison Burman/

Terry Jackson

Vaccinia virus

National Veterinary Institute

(Denmark)

Graham Belsham

University of Oxford

Elisabeth Fry/

David Stuart

structural biology

ConsortiumIAH-Immunology

Bryan Charleston

co-ordination

Sequences likely to enhance

expression in mammalian cells• At the 5’ end

→ not all the T7 transcripts get capped during co-expression of two recombinant vaccinia viruses

→ 75% initiation downstream of the FMDV IRES occurs at the second AUG

• At the 3’ end

3’UTR

+ poly (A) tail → separately enhance IRES mediated translation

L P2 P3P1VP3 VP1

2A

VP0 3C

3B3

3C

3B3

3'UTR 3'UTR

LbLa

IRESIRES

IRES

+ “La”

→ separately enhance IRES-mediated translation

Proof of principle that an engineered mutation (his to cys) is

consistent with capsid assembly.

Similar approaches can be used for infectious copies.

Structure-based stabilisation of FMDV capsids

Proof of principle that an engineered mutation (his to cys) is

consistent with capsid assembly.

Similar approaches can be used for infectious copies.

Structure-based stabilisation of FMDV capsids

Covalent Cage Particle

Characterisation

CC and WT empties were treated for 2h at 56ºC (or for

30min at pH5), then subjected to sucrose density

gradients.

6 7 8 10 11 12 6 7 8 10 11 12

175

83

62

47.5

32

25

16.5

WT CC

Fraction no. Fraction no.

Assembled empty

particles seen in CC

fractions only.

- Enhanced storage characteristics of formulated products

-Enhanced duration of immunity when combined with depot delivery system

-Improved T cell responses as a consequence of enhanced antigen presentation

Improved stability

Persistence of FMDV antigen- maintaining protective antibody responses

FMDV D46 DAPI

LZ

DZFMDV D46

80 µm

MLN 38DPCI

4 µmDAPI CD21 FMDV

No expression of non-structural

proteins:

-Non-replicating

-Extracellular

HypothesisIn the major wildlife reservoir of FDMV in Sub-Saharan Africa, the buffalo, depots of viable, non-replicating viruses

seed other tissue that results in the production of transmissible virus.

Primary objectives1: Determine whether FMDV capsid and genome are present in buffalo lymphoid tissue.

2: Determine whether secondary sites of FMDV localisation or replication are detectable in buffalo.

3: Determine whether infectious virus can be isolated from buffalo lymphoid tissue or epithelium after the

resolution of clinical signs.

4: Compare the genetic complexity of viral depots in buffalo tissue with virus present in oropharyngeal samples

collected by probang.

5: Develop minimally invasive sampling techniques for lymphoid tissue from buffalo to aid surveillance.

Collaboration with OVI in southern Africa