Download PPT 55

Localisation of FMDV after acute
infection in cattle;
a novel, immunologically
significant site
Nick Juleff
EuFMD 2008
Project background
FMDV:
• Vaccination (inactivated viral antigen)
–
–
•
Short duration of immunity
Antibody titres wane after several months
Natural infection
–
–
Prolonged virus-neutralising antibody titre and
immunity to infection (Cunliffe, 1964; Doel, 2005)
50% ruminants ‘persistently infected’. Viral excretion
is intermittent and declines progressively (Donn et al 1994;
Salt, 1993)
1. Mechanism maintaining elevated neutralising
IgG titres after infection?
2. ‘Persistent infection’ despite the high level of
neutralising antibody?
Hypothesis
•
Persisting virus (intact viral capsid) is required
to maintain neutralising antibody titres and
immunity following natural infection
•
Viable immune complexed FMDV is maintained
on Follicular Dendritic Cells (FDC) within
lymphoid tissue following natural infection
What are FDC?
Cardinal features
(They are not dendritic cells. Probably fibroblast derived)
Non-endocytic cells located in lymphoid follicles of secondary
lymphoid tissue (includes MALT).
• FDC bind and retain antigens in the form of immune complexes
(not internalised)
• Retained antigen is preserved and maintained in its native,
stable conformation for months. Reservoir of antigen. (Tew and Mendel,1979)
Figure 9-14
LZ
DZ
Bovine palatine tonsil GC
Not a homogenous population
Heterogeneity may relate to state of
activation
Light zone vs dark zone FDC
Is FMDV retained on FDC?
Examined:
• DSP – historical association with persistence
• RPLN and MLN – afferent lymph drainage
• Pharyngeal t and palatine t – sampled by probang
29 – 38 days post contact infection (O/UKG34/2001 and
O1BFS1860)
In situ hybridization
•DIG labelled RNA probes in combination with tyramide signal amplification
•Difficult technique, signal only detected associated with GC in lymphoid tissue
Mandibular lymph node (38 days post contact infection)
FMDV 3D antisense
Scale bar = 200 µm
IgG1 antisense
FMDV 3D sense
(SVD antisense)
© PLoS ONE 2008
Laser capture microdissection
• Six tissue sections per cap
• Samples processed in triplicate
Quantitative Real Time RT-PCR
• FMDV RNA standards (in duplicate)
• Bovine 28s rRNA standards (in duplicate)
© PLoS ONE 2008
CT values of GC samples 38.74 – 46.24
© PLoS ONE 2008
CT values of GC samples 36.76 – 40.22
© PLoS ONE 2008
© PLoS ONE 2008
CT values of GC samples 35.73 – 39.92
© PLoS ONE 2008
© PLoS ONE 2008
CT values of GC samples 34.68 – 37.01
© PLoS ONE 2008
CT values of GC samples 35.64 – 40.03
© PLoS ONE 2008
Germinal centre samples +ve by quantitative rRT-PCR
• n = 4, adjusted mean ± standard error of the mean (ANOVA, General Linear Model)
• Only germinal centre samples contained genome (after 50 cycles)
•10^8 copies 28s rRNA ≈ 100 cells (PBMC)
• Significant associated between quantity FMDV genome and tissue sampled
(p = 0.0039, Fisher’s Exact Test)
• MLN vs RPLN (p = 0.0014)
• MLN vs pharyngeal t (p = 0.0392)
• MLN vs palatine t (p = 0.0376)
• MLN vs DSP (p = 0.0148)
(ANOVA, Tukey simultaneous test)
© PLoS ONE 2008
Immunohistochemistry
• Dark zone
• Light zone
© Garland Science 2005
• Conformational, nonneutralising epitopes of FMDV
capsid
© PLoS ONE 2008
scale bar = 100 µm
Scale bar = 20 µm
© PLoS ONE 2008
50 µm
20 µm
© PLoS ONE 2008
• Extracellular?
4 µm
© Garland Science 2005
Immunohistochemical analysis of tissue 29 to 38 days
post contact infection for FMDV capsid and FMDV 3A
and 3C non structural proteins*.
Tissue
Number of animals
sampled
FMDV capsid +ve germinal
centres**
DSP
17
0
Pharyngeal tonsils
10
0
Palatine tonsils
10
6
RPLN
10
8
MLN
22
22
* Tissue was negative by immunohistochemical analysis for FMDV NSP.
** Number of animals with germinal centres staining positive for FMDV capsid.
DSP = dorsal soft palate.
RPLN = retropharyneal lymph node.
MLN = mandibular lymph node.
Of animals examined, five negative by probang
Conclusion from our studies
• FMDV is maintained in the light zone of GCs in lymphoid tissue
following natural routes of infection
• FMDV is maintained in association with FDCs (CNA.42)
• Non-replicating state (intact viral capsid and genome, no NSP)
Our data suggests:
– FMDV is maintained as immune complexes
– extracellular (desirable for a cytopathic virus)
– Intact viral capsid is essential for maintaining elevated protective
neutralising antibody titres
– Vaccine strategies – take capsid localisation and persistence into
account
• Immune complexed FMDV is able to infect FcR expressing cells ex
vivo and in vitro. We hypothesise that FMDV maintained on FDC is
viable. (macrophages, B cells, DCs?)
• This finding could explain FMDV persistence despite the high level
of neutralising antibody.
Thank you
Bryan Charleston
Zhidong Zhang
Ivan Morrison
Miriam Windsor
Liz Reid
Julian Seago
Haru Takamatsu
Lucy Robinson
Kerry Mclaughlin
Nigel Ferris
Animal work
Bartek Bankowski
Debi Gibson
Digital imaging
Paul Monaghan
Pippa Hawes
Jenny Simpson