病毒灭活血小板临床应用进展
邱艳
北京血液中心
2013年9月5日淮安
为什么病毒灭活成分血？
Bacteria
The most frequent
transfusion-transmitted infection
Known pathogens
New and emerging pathogens
Routine testing covers only
a limited number
A risk that current safety
measures cannot eliminate
Screening limitations
Leukocytes
Gaps in current defenses exist,
due to the window period and
limited screening sensitivity
Residual cells and cytokines
can cause harmful posttransfusion reactions
Most transfusion recipients are battling serious
disease and have weakened immune systems
Emerging Infections Disease: continue to emerge
worldwide
Red – newly emerging diseases: Blue – re-emerging/resurging diseases
Adapted from Morens DM. Nature. 2004;430:242–9.
Timeline of the WNV epidemic in the USA
PUBLIC HEALTH: WNV was recognized as a threat in 1999
First identification in
humans, horses and
birds in three states
1999
2000
BLOOD SAFETY:
Experimental tests
for WNV were not
implemented until
June 2003
66 human
cases reported
in 10 states
2001
9,862 human
cases, WNV Ab
test approved
2002
First documented
transmission by
organ transplant
and blood
transfusion
2003
WNV activity in
47 continental states,
2,470 human cases
2004
WNV NAT implemented
under IND; transmission
confirmed or suspected
in >40 blood recipients
2005
199 WNV-(+)
donors in
28 states
West Nile virus in Europe
 Recent outbreaks of WNV in Europe
reaffirmed that mosquito-borne viral
diseases may occur on a mass scale,
even in temperate climates1
 Frequent detection of equine, avian
and human WNV in south France
(2000 and 2003) suggests that some
areas may be at increased risk of
infection on a seasonal, rather than
sporadic, basis2
WNV isolation from mosquitoes
or vertebrates, including humans
Laboratory-confirmed human or
equine cases of West Nile fever
1. Hubálek Z, et al. Emerg Infect Dis. 1999;5:643–50.
2. Dauphin G, et al. Comp Immunol Microbiol Infect Dis. 2004;27:343–55.
Presence of antibodies
to WNV in vertebrates
Healthy blood donors can carry a range of
asymptomatic herpesviridae infections
Virus
Potential symptoms in
recipient*
Prevalence
Blood supply
screening
CMV
Pneumonitis, retinitis, birth
defects
50–85% by age 401
Tested as
needed
EBV
Mononucleosis, pneumonitis,
hepatitis, oncogenic potential
~95% by age 402
Not tested
HHV-6
Pneumonitis, fever,
encephalopathy, rash
>90% in adults3
Not tested
HHV-7
Pneumonitis, fever,
encephalopathy, rash
>90% in adults3
Not tested
HHV-8
Kaposi’s sarcoma
Varies by geography**
Not tested
*Severe symptoms most likely in immunocompromised individuals. **Moderate prevalence in
Mediterranean countries, low in rest of Europe.
1. http://www.cdc.gov/ncidod/diseases/cmv.htm 2. http://www.cdc.gov/ncidod/diseases/ebv.htm
3. Clark DA. Int J Hematol. 2002;76:246–52.
Studies have shown CMV transmission by
CMV-negative and leukoreduced blood products
Study
reference
Number of
patients
CMV
infection
CMV
disease
Type of blood
product used
Bowden, et al.
(1995)
252
250
2/4*
3/6*
0/0*
3/6*
CMV-negative
Leukoreduced
Ljungman, et al.
(2002)
33
49
3
6
0
0
Combination
Leukoreduced
360
6
Not given
447
18
Not given
CMV-negative or
leukoreduced
Apheresis platelets
Ronghe, et al.
(2002)
93
0
0
CMV-negative RBC
and leukoreduced platelets
Foot, et al.
(1998)
110
1
0
CMV-negative
Nichols, et al.
(2003)
*Primary and secondary endpoints (infections day 21–100 or 0–100 after SCT)
Adapted from Ljungman P. Br J Haematol. 2004;125:107–16.
Availability of safety measures against transfusiontransmitted infections
Anti-HCV
Syphilis
1960
HIV
p24
Anti-HIV-1
1965
1970
HBsAg
1975
1980
1985
NAT
1990
1995
Anti-HBc,
Anti-HTLV-I/II
Anti-HIV-2
Adapted from Barbara, J. Transfus Med Hemother. 2004;31(suppl 1):1–10.
2000
2005
Leukodepletion
Pathogen
Inactivation
血液制品病毒灭活的历史
IV Therapeutics
IV solutions
Plasma fractions
(FVIII, FIX,
albumin)
Plasma/FFP
Process
Availability
Sterilization
1950’s
SD(one or more
stages)
1980’s
SD, methylene blue
1990’s
血浆成分血病毒灭活的现状
Trade Name
Octaplas®
（SD）
and Method
solvant :Tri (n) butyl
phosphate (TnBP)
détergent :Octoxynol-9 or
Triton X 100 or Tween 80 or
Chlolate de sodium
核黄素+285365nmUVA+UVB照射
Mirasol
Theraflex©
MB-plasma
Intercept
Active Compound
®
1 mM Methylene blue(MB)
+180 J/cm2可见光照( 590 nm)
Amotosalen HCl（S-59)+
UVA light
Developer
B. HOROWITZ
(NYBC,1985 – 1990)VI
Technologies (Vitex)
ARC OctaPharma
Approval/
Phase
licensed in the US but
is withdrawn and
20130117 approved
for Blood-Clotting
Disorders in US
1992 approved in
France
2006 EU Mark
Navigant /
Caridian-BCT
CE mark in 2008
Springer，德国；
Grifols,西班牙
Use in 20 countries
MacoPharma 2008
approved in France.
Oct. 2011 withdrawn
in France
Baxter/Cerus
2010 EU Mark
regulatory review in
US
血小板成分血病毒灭活的现状
Trade Name
Active Compound
And Method
Developer
Approval/Phase
2002 CE Mark classe 3
INTERCEPT
Blood
System™
Amotosalen HCl （S-59）
+3 Joules /cm2 UV A
Baxter/Cerus
light (320-400 nm)
2005 Afssaps France
2007 PEI German
2010 wissmedicSwis
Phase III in US
Mirasol®
PRT
Theraflex©
UV
Riboflavin核黄素+ 6.2
J/cm2 UV /visible light
(265-370 nm)
0.3 J/cm2
monochromatic
UV light(200-275 nm )
Navigant /
CaridianBCT
CaridianBCT/
MacoPharma
Phase I/II/III
R&D
红细胞成分血病毒灭活的现状
Trade Name
Active Compound
and Method
Developer
Approval/
Phase
Phase I/II/III in US
stopped for
FRALE
S-303 (bifunctional
Baxter/Cerus
alkylator)
neoantigen
Phase I in EU
Phase I in US
INACTINE
PEN 110
Phase III
Navigant/
FDA approved
clinical trials in the
US
(Ethyleneimine)
Riboflavin(Vitamin B2)
Riboflavin
VI Technologies
2
+ 6.2 J/cm UV (265370 nm)
Caridian-BCT
血小板成分血病毒灭活的机理
X, G
Radio
WAVE LENGTH
(nm)
High energy
200
250
UVC
300
350
UVB
400
UVA
Amotosalen
INTERCEPT
3 J/cm2
Mirasol
6.2 J/mL
UVC
0.4 J/cm2
Low energy
590
750
VISIBLE
The INTERCEPT Blood System
Introduction & Mechanism
Nucleic acid targeting using amotosalen HCl
 Psoralen targets DNA and RNA
(single- and double-stranded)
 Crosslinking reaction is initiated
by UVA light (320-400nm)
NH2
O
 Replication of nucleic acid of
pathogens and leukocytes is
stopped
O
O
 Platelets, plasma and RBCs do
not require nucleic acid function
for therapeutic effect
Amotosalen
O
Amotosalen crosslinks both single- and
double-stranded nucleic acids
Helical
Regions
Double-stranded
DNA or RNA
Single-stranded
DNA or RNA
Amotosalen mechanism of action
UVA Illumination
Amotosalen
(S-59)
Targeting
Intercalation
Helical region of singleor double-stranded
DNA or RNA
Crosslinking
Multiple crosslinks
block strand separation
and replication
The INTERCEPT Blood System for platelets:
A broad spectrum of inactivation
 Bacteria
» Aerobic and anaerobic species
» Gram-positive and Gram-negative species
» Spirochetes (including Treponema pallidum and
Borrelia burgdorferi)
 Viruses
» Enveloped and non-enveloped species
» Species with either single- or double-stranded DNA or RNA
 Protozoa
» Including Plasmodium spp. (malaria), Trypanosoma cruzi
(Chagas’ disease), and Leishmania spp. (leishmaniasis)
 Residual donor leukocytes
» Prevents replication and inhibits cytokine synthesis
Limitations of the INTERCEPT Blood System
 Not effective against HAV
» Highly infrequent labile component pathogen with few
reported cases
 Not effective against bacterial spores
» Effective against vegetative state
 Not effective against prions
» Unique disease mechanism
» Very low frequency
» Preventable by other measures: livestock management
The INTERCEPT Blood System for
Platelets
Virus
The INTERCEPT Blood System for platelets:
Viral inactivation overview
Enveloped
ss RNA
HIV-1, HIV-2, HCV, HTLV-I, HTLV-II, BVDV, WNV
ss/ds DNA
HBV, DHBV
ds DNA
CMV
Non-enveloped
ds RNA
Bluetongue virus
ss RNA
Calicivirus
ds DNA
Simian virus 15
ss DNA
Parvovirus B19

Small non-enveloped picorna viruses (e.g., poliovirus, HAV) are
resistant to inactivation
Measurement of pathogen inactivation:
Log reduction
 灭活水平计算公式:
Log (Pre-treatment infectivity / Post-treatment infectivity)
或
Log (Pre-treatment infectivity) – Log (Post-treatment infectivity)
 处理后没有存活病毒,用 <1 infectious unit/volume assayed表示

“>” 表示灭活效果的最低检测限
 Log reduction was reported based on the volume assayed
Examples of log reduction calculations
Pretreatment
infectivity
(IU/ml)
Posttreatment
results
Posttreatment
infectivity
(IU/ml)
Log reduction
calculation
Log
reduction
106
5 organisms
in 1 ml
5
log106 – log5
5.3
106
5 organisms
in 10 ml
0.5
log106 – log0.5
6.3
106
No organisms
in 1 ml
<1
> (log106 – log1)
>6
106
No organisms
in 10 ml
<0.1
> (log106 – log0.1)
>7
INTERCEPT Blood System for platelets:
Inactivation of routinely tested pathogens
Pathogen
Log reduction*
Hepatitis B
• HBV (human, MS-2 strain)
• DHBV (model virus for HBV)
>5.5
>6.2
Hepatitis C
• HCV (human, Hutchison strain)
• BVDV (model virus for HCV)
>4.5
>6.0
HIV
• Cell-free HIV-1
• Cell-associated HIV-1
>6.2
>6.1
HTLV-I
• HTLV-I
• HTLV-II
4.7
5.1
Treponema pallidum (syphilis)
>6.8 to <7.0
*Approved product claims under CE Mark. “>” refers to below limit of detection for assay.
INTERCEPT Blood System for platelets:
Inactivation of additional enveloped viruses
Pathogen
Log reduction
Cytomegalovirus
>5.9*
SARS-CoV
>6.21
Vaccinia virus
>4.72
West Nile virus
>5.52
“>” refers to below limit of detection for assay. *Approved product claims under CE mark.
1. Pinna D, et al. Transfus Med. 2005;15:269–76. 2. Lin L, et al. Transfusion. 2005;45:580–90.
INTERCEPT Blood System for platelets:
Inactivation of non-enveloped viruses
Pathogen
Bluetongue virus, type 11
Parvovirus B191
• standard protocol
• 30 min incubation†
Log reduction*
6.1–6.4*
2.0
3.9
Feline conjunctivitis virus (calicivirus)
1.7–2.4*
Simian adenovirus 15
0.7–2.3*
Porcine parvovirus
0–0.2*
*Approved product claims under CE mark.
†This incubation period was supplementary to the standard operating protocol.
1. Sawyer L. Manuscript in preparation.
Implementation &
Data Management
INTERCEPT data management system (IDMS)
IDMS
server
HOST
system
Registration
Add amotosalen
Illumination
CAD incubation
Storage
Macopharma Platelets
谢谢!