Escape mutations in occult and reactivated hepatitis B virus - - PowerPoint PPT Presentation

Wolfram H. Gerlich Institute for Medical Virology

German Consulting Laboratory for Hepatitis B

AREVIR-GenaFor Meeting 10 April 2008 Stiftung caesar, Bonn

Escape mutations in occult and reactivated hepatitis B virus infection

Definition of occult HBV infection: serum HBsAg non-reactive in spite of proven (or suspected) HBV infection

• ften, but not always positive for HBV DNA

gold standard: liver tissue

Entry of hepatitis B virus, start of replication

immunity slow replication inapparent transient infection

Rapid immune response

HBs antigenaemia: too low or duration too short

Detection of transient occult HBV infection by single sample NAT

• Blood donor positive in Ultrio NAT

– 90 000 ge/mL HBV DNA – HBsAg negative: S/CO 0.6 in Prism – All HBV antibodies negative – ALT normal

• 32 days later

– ALT elevated – Anti-HBc and anti-HBs positive – HBV DNA positive, HBsAg negative

• 57 days later

– ALT normal, HBV DNA negative, anti-HBc/s positive

Communicated by P. Torres, Centro de Transfusion, Madrid and P.N. Lelie, Chiron

HBV from transient occult infection

• 90 000 ge/mL viral load, but HBsAg undetectable

– On average: HBsAg detectable if >2000 ge/mL

• Virus without HBsAg? No

– Normal density before and after detergent treatment – Immune precipitated by anti-preS1, but not by anti-HBc

• Mutated HBsAg? No

– No mutations in the HBsAg loop

• Low excess of subviral HBsAg particles ? Possibly

– Frameshift in the S ORF in one of 3 clones

• Rapid immune response ? Probably

– Precore stop mutant: HBeAg negative – Quasispecies – Mutations in preS, in S/rt and in X – e. g. rt119, 149, 153, 213, 237, 257, 267

Entry of hepatitis virus, start of replication

acute hepatitis

immunity recovery slow replication

strong replication

inapparent transient infection

Delayed immune response

immune defence

HBsAg positive

Anti-HBs HBsAg

HBV DNA PCR

Anti-HBc

Acute resolving hepatitis B

disease months

Recovery Occult

Transmission of HBV by blood from donors with completely occult early phase infection

Three independent cases from Germany

• Multiple donors recognised by seroconversion to

HBsAg positive: look back

• Previous donations had been missed by minipool

testing for HBV DNA

• Retesting of stored single samples with realtime

PCR, detection limit < 12.5 IU/mL negative

• Recipients of red cell concentrates: infected,

HBsAg +, HBV DNA +, anti-HBc -, same sequence, wildtype virus

Anti-HBs HBsAg

HBV DNA PCR

Anti-HBc

Acute resolving hepatitis B

disease months

Recovery Occult ?

Anti-HBs HBsAg

HBV DNA PCR

Anti-HBc

Acute resolving hepatitis B

disease months

Recovery Occult, low level persistence

Types of occult HBV infection: HBsAg non-reactive, but infected

• Transient inapparent infection

– Seemingly occult during normal acute infection

• Early window period
• Late acute window period
• Low level persistence in occult carriers after

– Acute „resolving“ hepatitis B – Inapparent infection

Course of hepatitis B virus infection

acute fulminant immunity recovery weak replication strong replication inapparent infection Delayed, vigorous immune response immune defence

Occult intrahepatic persistence

Infectious blood and liver donations

Anti-HBs HBsAg

HBV DNA PCR

Anti-HBc

Acute resolving hepatitis B

disease months

Occult, low level persistance

Escape mutants

HBV escape mutants

• Anti-HBs blocks intercellular spread of HBV

– can not stop intracellular pre-existing virus

• HBV replication is inaccurate
• Mutated HBsAg escapes recognition
• Mutated HBV can spread within liver

G145R

Model of the HBsAg loop (aa 98 – 161): typical escape mutation after vaccination of HBV infected newborns

a-determinant Carman et al., Lancet 1992;336:325 mutation

HBV transmissions by occult infected blood donors

Summary of data from Germany and Denmark 2004 - 2005

• 5 donors

– HBV DNA 9 – 240 ge/mL, no anti-HBs – In four donors several mutations of the HBs loop – Only one donor had wildtype HBsAg

• 55 recipients, 68% anti-HBc pos.

– 10 possible transmissions, asymptomatic – 22 probable transmissions, asymptomatic – 3 fatal hepatitis B cases (donors I – III)

• cofactors: immunosuppression, sepsis

– no normal acute hepatitis B after transfusion

• W. Gerlich et al. J Med Virol 2007; 79:S32-S36

G145K L

Mutations in occult infected blood donor

Fatal transmission I, 240 ge/mL, 14 clones sequenced

E

L T

a-determinant C121-C148

L

I N F

Mutations in anti-HBc+/s- infectious blood donor

Fatal transmission II, ca. 50 ge/mL

HBsAg subtype change a-determinant C121-C148

No mutations in anti-HBc+/s- infectious blood donor

Fatal transmission III, ca. 10 ge/mL

a-determinant C121-C148

Occult infected blood donor IV

• Ca 2000 IU/mL (10 000 ge/mL) HBV DNA

– HBsAg in Abbott‘s Prism negative, <0.1 IU/ mL – Anti-HBc weakly positive, S/CO 0.4 – IgM anti-HBc negative

• Anti-HBs negative, but
• HBsAg loop mutated, quasispecies

– Direct sequencing and 10 cloned sequences: – 5 of 6 mutations not detected by direct sequencing

Communicated by M. Schmidt Red Cross BDS, Frankfurt

I I

Model of the HBsAg loop IV

S

Mutations in anti-HBc+/s-, HBV DNA pos. blood donor IV H

N A heterogeneous 2000 IU/mL 10 clones sequenced

a-determinant C121-C148

Anti-HBc only : donor related look back

• Anti-HBc screening in Germany since 2006
• One long-time blood donor has anti-HBc, no anti-HBs
• HBV DNA in 5 of 5 consecutive donations pos.: 9 to 46 ge/mL
• 5/13 red cell recipients anti-HBc positive
• 11/11 recipients of fresh frozen plasma anti-HBc positive

– probably all plasma donations infectious, – but no hepatitis

• numerous mutations in the HBsAg loop
• dynamic quasispecies
• A. Lattermann, Lahn-Dill-Klinik Wetzlar, S. Wienzek, et al., Institute of Clinical

Immunology and Transfusion Medicine, Giessen, Germany

I H

Model of the HBsAg loop IV

Q F

Mutations in anti-HBs negative, infectious blood donor V

1st sample, 23 ge/mL, 10 clones sequenced K N S V

homogeneous heterogeneous

I H

Model of the HBsAg loop IV

Q F

Mutations in anti-HBs negative, infectious blood donor V

2nd sample, 9 weeks later, 9 ge/mL, 7 clones K N S S V transient

I H

Model of the HBsAg loop IV

Q F

Mutations in anti-HBs negative, infectious blood donor V

3rd sample, 29 weeks later, 34 ge/mL, 12 clones K N S R

I H

Model of the HBsAg loop IV

Q F

Mutations in anti-HBs negative, infectious blood donor V

4th sample, 38 weeks later, 46 ge/mL, 10 clones V P G K N S R,stop

Pseudo-occult HBV infection in blood donor VI

• Negative in Ortho‘s HBsAg screening assay
• Strongly positive in Abbott‘s HBsAg assay
• 24 IU/L anti-HBs and anti-HBc-IgG
• 200 genomes /mL HBV DNA
• Direct sequencing: escape mutations

– 1st sample: L109V; M133T; D144G

• Look back

– 3 recipients not infected, 1 anti-HBc/s positive

Communicated by Drs. Ullum and Dickmeiss, Copenhagen, DK

HBV escape mutations in blood donor VI

• Negative in Ortho‘s HBsAg screening assay
• Strongly positive in Abbott‘s HBsAg assay
• 24 IU/L anti-HBs and anti-HBc-IgG
• 200 genomes /mL HBV DNA
• Direct sequencing: escape mutations

– 1st : L109V…………………………………………………M133T D144G – 2nd : L109V P120L C121Y P127T G130D M133T

• Look back

– 3 recipients not infected, 1 anti-HBc/s positive

Communicated by Drs. Ullum and Dickmeiss, Copenhagen, DK

V,Q

Model of the HBsAg loop IV

Insertion T C122Y D

Escape mutations in HBsAg and anti-HBs pos. blood donor VI

2nd sample, 240 ge/mL, 19 clones sequenced

P127T G A

stop

R P120L T heterogeneous

T

Aa-sequence of S-loop

Donor VI, immuno- selection

M133T

wt

19 mutant clones 13 different sequences

Sequence of HBs loop

Aa-sequence of S-loop

HBV carrier, immuno- tolerant

Sequence of HBs loop

9x M1R 3x M1L 1x M1G 1x M1K

Elimination of the start codon (M)for the small HBs protein

Donor VI, immune selection

3x M wt

Donor V 1

100 200 300 400

preS1 preS2 SHBs

2

100 200 300 400

3

100 200 300 400

4

100 200 300 400 300

5

100 200 400 300

Donor I

100 200 400 300

Donor IV

100 200 400 300

Donor VI

100 200 400 300

reactivated

100 200 400 300

TM 1 TM 2 Internal loop Envelopment HBsAg loop Variable topology a- determinant Attachment

homogeneous heterogeneous mutation

Variability of S ORF and major neutralising epitopes in 4 blood donors with (pseudo-) occult HBV infection

Variation % a

Type of HBV infection Ge/mL S ORF HBs loop b preS1 109 2000 240 23 9 29 38 200 Anti-HBc+/s- 63/4000 1,6% 4.4% 0.3% 0.6% 2.0% 1.0% 2.2% 1.9% 2.9% 2.5% Anti-HBc+/s- 1 164/4000 4.1% 10.3% Donor C, 2 94/2800 3.4% 12.9% Donor C, 3 168/4000 3.5% 10.5% Donor C, 4 176/4000 4.4% 14.4% HBs/eAg+, control 25/12400 0.2% 0.2% Anti-HBc+/s- 287/7600 3.6% 8.7% HBsAg-c /+ Anti-HBc+/s+ 270/14400 3.8% 5.9% Ne/Nt a

a) number of exchanged aa/number of sequenced aa positions in 7 to 36 clones from amplified S ORF b)Aminoacid 99-170 of SHBs c) negative in screening test

• Variation 8-22 times higher than in immunotolerant carriers
• HBsAg loop is 22 to 72 times more variable
• probably driven by neutralising antibodies

– but anti-HBs often undetectable

• More and other mutations

– than after vaccine or HBIG induced escape. – often upstream or downstream of determinant „a“ – mutations of subtype determinants d, y, w1-4, r

• Major mutants are viable, infectious, and stable
• Neutralising preS1 epitopes more stable
• PreS1 antigen - a better target for diagnosis and vaccines?

Variability of S open reading frame (ORF) in

• ccult HBV infections: conclusions

Course of hepatitis B virus infection

acute fulminant immunity recovery weak replication

Strong replication

inapparent infection

Immune reconstitution

immune defence

Occult intrahepatic infection

Immunosuppression Reactivation

HBV Reactivation

• Possible in

– anti-HBc positive – or only anti-HBs positive – or seronegative persons

• Frequent in lymphoma/leukemia therapy
• Asymptomatic replication to high viremia

– under immunosuppression

• Immune reconstitution:

– Hepatitis

Immune suppression before under/after anti-HBc/s HBsAga PreS1c Liver transplant + / +d + / - +e 5 Stem cell therapy + / + + / + + 1 leukemia (+ / +) + / + + / − 10 2 lymphoma + / + + / − − / − 16 2

• Nr. of escape

mutations anti-HBc/s HBs loopb kidney transplant + / + + / − + / − 3 lymphoma + / + + / + + / − 5 lymphoma − / +f − / + + / − 3 lymphoma 1st lymphoma 2nd − / +f − / − − / − + / − + / − 5 5 2 2 Bone marrow transpl. + / + − / − + / +

Cause of immune suppression

Reactivated occult HBV infections

Serology and escape mutations in 9 cases

a HBsAg AxSym / or other tests

b HBsAg loop aa 99-170 c attachment site aa 20-59 d from HBIG e breakthrough after 10 years f no vaccination known

Unnoticed asymptomatic reactivation of HBV escape mutant in a dialysis patient

• Kidney transplant 10 years ago
• HBsAg screening negative (Enzygnost)
• Accidentially retested in AxSym: clearly HBsAg positive
• Viremia 108 HBV DNA molecules /mL
• Five S gene mutations
• Patient was anti-HBc,anti-HBs pos. before transplantation
• Reactivated escape mutant since 10 years

case reported by Dr Sabine Sussitz, LKH Klagenfurt, Austria

Escape mutations in asymptomatic reactivated occult infection after kidney transplantation

P120Q R L

a-determinant C121-C148

Acute (de novo?) infection with HBV escape mutant

• 5 fold elevated ALT
• Anti-HBc IgM S/CO 33
• HBsAg confirmed positive
• 2x108 ge/mL HBV DNA
• 260 IU/L anti-HBs, vaccination status unknown
• Several travels to high endemicity areas
• Transmission of an escape mutant?

Case communicated by M. Kiehntopf, R. Siegmund, University of Jena

Reactivated „acute“ infection with HBV escape mutant in anti-HBs positive leukemia patient

• Chronic leukemia
• 5 fold elevated ALT
• HBsAg confirmed positive
• Anti-HBc IgM S/CO 33
• 2x108 ge/mL HBV DNA
• 260 IU/L anti-HBs
• Several travels to high endemicity areas
• Genotype A2 of Central Europe
• 14 mutations in S Gene

Escape mutations in acute reactivated occult infection

T L N R R L T T R Subtype change HBsAg and anti-HBs pos., 108 ge/mL, 11 clones a-determinant C121-C148

Reactivation of HBV and acute hepatitis after lymphoma therapy

• Anti-HBs, anti-HBc positive before chemotherapy
• After therapy

–

rising ALT >2000 U/L – anti-HBs negative, anti-HBc positive

• 260,000 /mL HBV DNA, HBeAg positive
• HBsAg negative in Abbott‘s AxSym
• 23 mutations in 135 aa of S gene

Case communicated by C. Rompf, Dortmund

Reactivated severe hepatitis B after lymphoma therapy

2000 U/L ALT, 64 000 ge/mL, 11 clones sequenced Y C I L I P I R E L T F H D144G S

Mutations

T

I K

HBV reactivation (?) in a lymphoma patient

Communicated by P. Schnitzler, University of Heidelberg Rituximab therapy 1010 ge/ml HBV DNA Anti-HBc/s negative a-determinant C121-C148

D144A I

R K S L

Fatal HBV reactivation in a lymphoma patient

Westhoff et al., Blood 2003; 102:1930

109 ge/ml HBV DNA Four of 7 HBsAg assays false negative a-determinant C121-C148

D144A

Clinically silent reactivation in lymphoma patient Anti-HBs positive, no anti-HBc before therapy

• 1989: B cell lymphoma, chemotherapies
• 1996: HBsAg neg., anti-HBc neg., anti-HBs 612 IU/L
• no vaccination known
• 2003: again chemotherapy

– HBsAg AxSym highly positive, not inhibitable – HBsAg VIDAS negative – Anti-HBc negative, anti-HBs 93 IU/L – No hepatitis – 1.8x108 ge/mL HBV DNA

• Reactivation of HBV, 3 escape mutations
• S. Awerkiew et al., J Clin Virol 2007, 38:83-86; University of Cologne

R C137W G145R

Clinically silent reactivation in unvaccinated lymphoma patient anti-HBs positive, no anti-HBc before therapy

• S. Awerkiew et al., J Clin Virol 2007, 38:83-86

a-determinant C121-C148

T N G145R

Clinically silent reactivation in unvaccinated lymphoma patient Anti-HBs positive, no anti-HBc before therapy II

Communicated by J. Verheyen, University of Cologne Under chemotherapy: 1st sample HBsAg + Anti-HBs - Anti-HBc - 5x108 ge/mL 25 clones heterogeneous a-determinant C121-C148

L

N G145R

Clinically silent reactivation in unvaccinated lymphoma patient Anti-HBs positive, no anti-HBc before therapy II

Communicated by J. Verheyen, University of Cologne 2x108 ge/mL 2nd sample 8 weeks later a-determinant C121-C148

Summary: Occult persistent HBV infections

• develop after inapparent or apparent hepatitis B
• HBsAg is nonreactive because of

– Too low amount or – Escape mutations – Or both

• HBV DNA may be undetectable in serum

– Intrahepatic reservoir

• Anti-HBc may be absent
• Anti-HBs may be the only marker
• Significant, because

– HBV may be transmitted via blood or liver donations – may reactivate to high infectivity and pathogenicity

Summary: Reactivation of occult HBV infection and its prevention

• Induced by severe immune suppression
• Clinically silent under immune suppression

– High infectivity

• Mutations in the HBs loop frequent

– Escape from endogenous or vaccine-induced antibodies – False negative HBsAg results

• Highly pathogenic under immune reconstitution

– Therapy often comes too late

• Reliable early detection necessary
• Before immune suppression

– Testing for HBsAg, anti-HBc and anti-HBs

• Monitoring of patients

– with HBV DNA realtime PCR

Dieter Glebe Mona Saniewski Christian Schüttler Ulrike Wend Wulf Willems

Many thanks to

Supported by DFG Collaborative Research Centre SFB 535 Project A2 and WHO/PEI