Update on CDC XMRV Activities

R. Michael Hendry, D.Sc.Chief, Laboratory Branch

DHAP, NCHHSTP, CDC

Blood Products Advisory CommitteeJuly 26, 2010

The findings and conclusions in this presentation are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention

CDC Activities: Retrovirology, CFS, and Blood Safety

• Retrovirology:

Laboratory Branch

Division of HIV/AIDS Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention

• CFS, Epidemiology:

Chronic Viral Diseases Branch

Division of High-Consequence Pathogens and Pathology

National Center for Emerging and Zoonotic Infectious Diseases• Blood and Tissue Safety:

Office of Blood, Organ, and other Tissue Safety

Division of Healthcare Quality Promotion

National Center for Emerging and Zoonotic Infectious Diseases

• High prevalence:Lombardi et al. 2009 - 68/101 (67%) CFS; 5/218 (3.6%) healthy persons (US)

- RNA PCR from plasma, proviral PCR, Flow-based antibody testing, culture (activated PBMCs, plasma)

• Zero Prevalence:Erlwein et al. 2010 - 0/186 CFS from UK

- proviral nested PCR; gag and pol)

Groom et al. 2010 - 0/170 CFS and 0/395 controls from UK - DNA PCR (gag and env) - 1/565 showed neutralizing activity (CFS patient), but was

nonspecific

van Kuppeveld et al. 2010 - 0/32 CFS and 0/43 controls from the Netherlands- DNA RT-PCR (int) and nested PCR (gag)- cDNA step first to increase assay sensitivity

Discordant XMRV Prevalence in Persons with CFS

Methods• Developed WB assay for antibody detection using polytropic MuLV-infected

(PMLV) and uninfected HeLa cells

- same assay format used successfully to identify human infection with simian foamy virus- plasma tested at 1:50 dilution

• Developed highly sensitive and specific nested PCR assays in multiple viral genes (gag, pol)

- samples screened with nested gag and pol PCR tests using 1 ug DNA input (integrity confirmed by B-actin PCR)

• Developed sensitive mouse sequence specific qPCR to detect contamination with mouse DNA

- XMRV positive DNA samples tested for mouse contamination

Strong Reactivity of MuLV antiserum to WB Antigen, CDCH

eLa/

XM

LVH

eLa

10080

605040

30

20

α Friend MuLV (whole virus)

250

500

1000

4000

2000

8000

16,0

0032

,000

α G

aLV

(p

30;

1:50

)

64,0

00P

re-i

mm

un

e

α Rauscher MuLV (gp69/71)

250

500

1000

4000

2000

8000

16,0

0032

,000

64,0

00P

re-i

mm

un

e

250

500

1000

4000

2000

8000

16,0

0032

,000

α M

uLV

wh

oe

viru

s

64,0

00

Pre

-im

mu

ne

α R

a M

uLV

En

vα XMRV (whole virus)

p30

gp69/71pr65

p30(CA)

p15E(TM)p15(MA)

gp69/71(Env)pr65(Gag)

Rat α SSFV Env (32,000), not shown

High Sensitivity and Specificity of PCR Assays, CDC

Gene Sensitivity2 Specificity Notes

XGAGN1

10 copies (34/34, 100%) 0/41 US BD (100%) Urismann et al.

XPOLN 10 copies (32/34, 94.1%) 0/41 US BD (100%) generic

MCOX2 10 copies (12/12, 100%) 0/117 US BD (100%) 1 cell equivalent

5 copies (12/12, 90%)

Proviral

Mouse-specific

Test

1. N, nested PCR2. Sensitivity determined using XMRV VP62 plasmid diluted in 1 ug human DNA or VP62 RNA

Rare XMRV Infection in Prostate Cancer, CDC

1. Percentages in parentheses2. Dashes indicate test not performed on these sample types3. nPCR, nested PCR

Sample Type Total XGAG XPOL MCOX2 WB

Prostate DNA 162 1(0.6) 3(1.9) 0/3 (0) -

Plasma 162 - - - 0/162 (0)

XMRV nPCR3

MurinePCR Serology

Switzer, et al. CROI, 2010

Study Population

• Archived, anonymous plasma and matching PBMCs/DNA from 51 persons with CFS and 56 matched healthy controls available for testing

• CFS defined using 1994 International Research Case Definition

• Population based (telephone interviews and clinical evaluation):- 11 CFS and 26 healthy controls from Wichita, KS- 22 CFS and 30 healthy controls from Georgia (rural, urban, metro)- 3/33 CFS (9%) reported sudden onset

• Physician referred CFS persons from Bibb County, GA with clinical evaluation:- 18 DNA- 19 plasma- included three persons (17%) with sudden onset

Lab Testing• Blood specimens tested using a combination of molecular and serologic

assays• Blinded testing and included positive and negative controls

at independent labs

• WB at CDC using a polytropic MuLV as antigen and comparison of reactivityto uninfected antigen to determine viral-specific seroreactivity

• Nested PCR to detect two gene regions at CDC:gag = XMRV specific but can detect polytropic MuLVpolymerase (pol) = generic for xenotropic and polytropic MulV- 10 copies/ 1 ug DNA sensitivity of each assay

• XMRV EIA and IFA at Robert Koch Institute (RKI), Berlin, Germany using recombinant XMRV Env and Gag proteins

• Nested gag PCR at Blood Systems Research Institute (BSRI), San Francisco, CA

- 3 copies/250 ng DNA assay sensitivity

Absence of XMRV in CFS and Healthy Persons from the US

Specimens CDC pol PCR

CDC gag PCR

CDC WB RKI EIA RKI IFA BSRI gag PCR

CFS 0/50 0/50 0/51 1/51 0/1 0/50

Healthy Controls 0/56 0/56 0/53 1/53 0/1 0/56

Absence of XMRV Antibodies in Additional Populations Tested at CDC

0/13 HTLV-1/2 +

0/7 HIV-1 +

0/6 HIV-1/HIV-2 dual +

0/121 US Blood Donors

0/20 US IVDU

0/20 “positive” plasmas from WPI

Pre

-im

mu

ne

α R

a M

uLV

(1

:50

0)

α F

r M

uLV

(1

:50

0)

1 2 3 4 5 6 7 8 9 10 11 12

100/12080605040

30

200

20

100/12080605040

30

200

20

p30(CA)

gp69/71(Env)pr68(Gag)

InfectedHeLa

UninfectedHeLa

Absence of XMRV antibodies in CFS patients by Western blot analysis, CDC

CFS

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

G17

G19

G20

G21

G22

G23

G24

G25

G26

G27

G28

G29

G30

G31

G32

G33

G34

G35

G36

G37

G38

G39

G40

G41

G42

G44

G45

G46

G47

G48

G49

G50

G58

G59

G60

G61

G62

G63

G64

G65

G66

G67

G68

G69

G70

G71

G72

G73

G74

G75

Mo

use

ser

a

W1

W3

W4

W6

W7

W9

W10

W11

W12

W13

W14

W15

W16

W17

W18

W19

W20

W21

W22

W23

W25

W26

W27

W28

W29

W30

W31

W32

W33

W34

W35

W36

W37 G1

G2

G3

G4

G5

G6

G7

G8

G9

G10 G11

G12

G13

G14

G15

G16

Mo

use

ser

a

0

0.1

0.2

0.3

0.4

0.6

0.7

0.8

0.5

OD CFSAssay cut-off

OD healthyOD positive control

OD

49

2/6

20

OD

49

2/6

20

Absence of XMRV antibodies in CFS patients and healthy persons by ELISA using XMRV rec-proteins, RKI

Absence of XMRV polymerase sequences in CFS patients, CDC

1° PCR

2° PCR

ß-actin

XM

RV

10

cop

ies

XM

RV

103

cop

ies

Neg

DN

AH

2OH

2O

CFS

10-1 to 10-4 BD DNAH

2OCFS

Absence of XMRV gag sequences in CFS patients, BSRI

GAPDH

2° PCR

XM

RV

10

cop

ies

XM

RV

3 c

op

ies

Neg

DN

A

H2O

XM

RV

1 c

op

ies

CFS

Absence of XMRV DNA in Additional Populations

• Kunstman et al. 2010, AIDS • 0/996 men from the Chicago MACS (562 HIV+, 434

HIV-) • proviral qPCR; gag (Singh primers)

• Gao et al. 2010, ICEID – Gen-Probe and ARC• 0/1435 blood donors from ARC, NC• 0/44 HIV-1+ blood donors• rtTMA; DNA and RNA

Conclusions and Summary• Developed highly sensitive assays for detection of human infection with

XMRV and other MuLVs

• We did not find any evidence of infection with XMRV in our study population of CFS patients and matched healthy controls

• PCR and serologic methods performed independently in three laboratories blinded to the clinical status of the study participants

• Testing included generic PCR and two serology assays which reducespossibility of false negative results caused by divergent viruses

• Differences in patient population, complexities of CFS, lab methods used, etc.may explain the contrasting results of our study and those of Lombardi et al.

• However, our results do not support an association of XMRV with the majorityof CFS cases

• More research is needed to determine the prevalence of XMRV in the generalpopulation, to investigate its transmissibility, and to standardize testing across labs

• More studies are needed to better understand the prevalence and significanceof XMRV in CFS, blood donors, and the general population.

Bill Switzer, Hongwei Jia, Shoahua Tang, Hao Zheng, Anupama Shankar, Bill Reeves, Rebecca Falkenberg, Walid Heneine

Acknowledgements

The findings and conclusions in this presentation are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The findings and conclusions in this presentation are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Robert Silverman

Sandy Ruscetti

Ila Singh

Oliver Hohn and Norbert Bannert

Graham Simmons