1. HBV/HIV co-infections: molecular and biological characterization of occult HBV strains or strains resistant to antiviral treatment
ANRS N°12149
Selma de ANDRADE GOMES
Lab. de Virologia Molecular
IOC-FIOCRUZ
Alan KAY
Centre de Recherche en Cancérologie de Lyon (INSERM U1052)
ANRS/Programa Nacional DST/AIDS Evaluation Meeting, Rio de Janeiro, 4-5 April 2011

2. History of the collaboration
In the early 2000s, our laboratory in Lyon was actively working on the problem of occult Hepatitis B Virus (HBV) infections:
Chemin, I., Zoulim, F., Merle, P., Arkhis, A., Chevallier, M., Kay, A., Cova, L., Chevallier, P., Mandrand, B., Trepo, C.,
2001. High incidence of hepatitis B infections among chronic hepatitis cases of unknown aetiology. J Hepatol 34,
Chemin, I., Jeantet, D., Kay, A., Trepo, C., Role of silent hepatitis B virus in chronic hepatitis B surface antigen(-)
liver disease. Antiviral Res 52,
 Jeantet, D., Chemin, I., Mandrand, B., Zoulim, F., Trepo, C., Kay, A., Characterization of two hepatitis B virus
populations isolated from a hepatitis B surface antigen-negative patient. Hepatology 35,
In Rio de Janeiro at the same time, Selma GOMES was also working on occult HBV infections, and more specifically in HIV co-infected patients:
 Gomes, S.A., Yoshida, C.F., Niel, C., Detection of hepatitis B virus DNA in hepatitis B surface antigen-negative
serum by polymerase chain reaction: evaluation of different primer pairs and conditions. Acta Virol 40,
 Santos, E.A., Yoshida, C.F., Rolla, V.C., Mendes, J.M., Vieira, I.F., Arabe, J., Gomes, S.A., Frequent occult
hepatitis B virus infection in patients infected with human immunodeficiency virus type 1. Eur J Clin Microbiol
Infect Dis 22, 92-98
 Santos, E.A., Sucupira, M.V., Arabe, J., Gomes, S.A., Hepatitis B virus variants in an HIV-HBV co-infected
patient at different periods of antiretroviral treatment with and without lamivudine. BMC Infect Dis 4, 29
Selma contacted our laboratory, and I visited Selma’s lab in 2005
We decided to apply for an INSERM/FIOCRUZ exchange agreement

3. HBV infection 350 million chronic HBV patients
> 10 times the number of HIV patients
 8%: High
2-7%: Intermediate
Brazil – Intermediate to high
< 2%: Low

4. Geographic distribution of HBV genotypes
Pujol and Devesa (2005) J Clin Gastroenterol. 39:611-8.

5. What is occult HBV infection?
HBV DNA
Acute infection: rapid loss (<6 months) of surface antigen (HBsAg) and HBV DNA
Chronic infection: persistence (>6 months) of HBsAg, persistence of HBV DNA
Occult HBV infection: detectable HBV DNA, HBsAg undetectable
 DNA levels usually very low (<1000 copies/ml)
 the virus can be transmitted
 retains the oncogenic potential of chronic HBV infections

6. Occult HBV infection in the literature
over years
More
Than 240
Number of publications
140
120
100 80 60 40 20
Chemin & Trepo J Clin Virol 2005, Adapted
Raimondo et al, J Hepatol 2008

7. Occult HBV and HIV co-infection
Study
Country
N° patients
Occult HBV
N° (%)
Hofer, 1998
Torres-Baranda, 2006
Filippini, 2006
Mphahlele, 2006
Pogany, 2005
Neau, 2005
Santos, 2003
Wagner, 2004
Goncales, 2003
Nunez, 2002
Piroth, 2000
Raffa, 2007
Switzerland
Mexico
Italy
South Africa
Netherlands
France
Brazil
Spain 57 35 86
140 93
160
101 30
159 85 37
51 (89%)
7 (20%)
17 (20%)
31 (22.%)
4 (4%)
1 (0.6%)
11 (37%)
16 (16%)
8 (5%)
13 (35%)
42 (41%)
Methods
“nested” PCR
(serial evaluation)
“nested” PCR (liver)
single step PCR
Cobas Amplicor HBV
Monitor (Roche)
Raimondo et al, J Hepaol 2007, modified

8. In Vitro Complementation and Ligation
Rolling circle amplification (RCA)
A new tool for amplifying full-length HBV genomes by completion and ligation of HBV RC-DNA to cccDNA
T4 DNA polymerase +
T4 DNA ligase
Hybridization
minus-strand primers
DR1
DR1
DR2
DR2
HBV RC-DNA
In Vitro Complementation and Ligation
HBV ccc-DNA
Elongation
minus-strand DNA
Strand
Displacement
Hybridization
plus-strand primers
End product –
High molecular weight
double-stranded DNA d.

9. Illustration of RCA Agarose gel of RCA products
M H2O
Copies / reaction
Agarose gel of RCA products
Southern blot of gel – HBV has been
succesfully amplified
Unit-length HBV genomes can be
excised from RCA products – can
be dirctly cloned and/or sequenced
Sensitivity can be increased by doing
A genomic PCR on RCA products
10 kb
3 kb
HBV genome (3.2 kb)
3 kb
HBV genome (3.2 kb)

10. 1) Reactivation of an occult HBV infection in a HIV+ patient
Applications
1) Reactivation of an occult HBV infection in a HIV+ patient
Patient HIV+, anti-HBc+, anti-HBs+ - profile resolved acute HBV infection?
Reactivated occult HBV infection after glucocorticoid treatment
Amplified, cloned and sequenced full-length HBV genomes after RCA
Genomes are of subgenotype A2 (European genotype A)
No drug resistance mutations despite poor compliance of patient
Genomes viable – replication competent, secrete viral particles
What causes immune-escape from anti-HBs?
● all genomes contain 2 significant mutations in the S gene
● K122R – very rare in genotype A
● D144E – potential immune-escape mutant

11. Seroconversion to a-HBe
What can the patient’s serum recognize?
Genetically engineered genomes so that they express viral particles of
wild-type HBsAg sequence, with K122R, D144E or both
Transfected into cells, labeled with 35S-Met/Cys, immuno-precipitated
secreted viral particles
K122/D144
K122R/D144
K122/D144E
K122R/D144E
C- C+ P
C- C+ P
C- C+ P
C- C+ P
C- = Normal human serum
C+ = Serum from a vaccinated person
P = Patient’s serum
HBsAg
Acute
Infection
< 6 Mo
Emergence of
K122R.
Low-level
HBV replication
HIV+
Wild-type
Virus
K122/D144
Occult
> 12 Yrs
Glucocorticoid
treatment
6 Mo Mo
Stimulation of
HBV replication.
D144E
Reactivation
2 Mo
K122R/D144E
Antiviral treatment
Seroconversion to a-HBe
HBV-

12. 2) Mutations associated with HBeAg-negativity in subgenotype F2
Applications
2) Mutations associated with HBeAg-negativity in subgenotype F2
prevalent in Brazil
HBeAg-negative mutants – important global health problem
Stop codon position 28 of precore region - not possible for genotype A or F2
Anna KRAMVIS – subgenotype A1 – mutations in core promoter/precore region
1762
1764
1809
1812
1814
1858
1862
1896
1901 A G
GCAC
ATG T G
TGG
ATG
Precore
HBcAg T A T T C T A
Core promoter
Genotype A
Subgenotype F2
HBV RC-DNA
DR1
DR2
1901
1762
Spe I
RCA
Spe I
1762
Full-length HBV genome

13. Amplification and sequencing of genotype F genomes
H20
RCA cut Spe I
HBV full-legth genome
RCA then genomic
PCR using primers
at Spe I site
HBV full-legth genome
#1 GGGGGAGGAGATTAGGTTAAAGGTCTTTGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGGCATGGAC
#2 GGGGGAGGAGATTAGGTTAAAGGTCTTTGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGGCATGGAC
#3 GGGKGAGGAGASTAGGTTAATGGTTTATGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGACATGGAC
#4 GGGKGAGGAGASTAGGTTAATGGTTTATGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGACATGGAC
F2 GGGGGAGGAGATTAGGTTAAAGGTCTTTGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGGCATGGAC
1762
1764
1809
1812
1814
1858
1862
1896
1901
Precore
HBcAg
Subtype F2 genomes do not show the mutations found with subtype A1
Some isolates (#3 & 4) have A1762T but not G1764A
Isolates #3 & 4 have other mutations before and after 1762/1764
Also have mutated codon 29 (GGC  GAC, Glycine  Tyrosine)
More work needed (sequence complete genomes, study HBeAg expression)

14. HBV-HIV co-infection
HIV interferes with the natural history of HBV infection by enhancing HBV replication, leading to more severe liver disease, decreasing hepatitis B ‘e’ antigen seroconversion and increasing HBV DNA levels
Due to the shared modes of transmission of HBV and HIV, the prevalence of HBV infected patients is higher than in non-HIV infected individuals
Occult HBV infection = Detection of HBV DNA in absence of HBsAg
Possible explanations: Low rate of HBV replication, mutations that inhibit HBsAg expression or change HBsAg antigenicity, thus preventing detection by commercial assays.
Among HIV-infected cohorts, occult HBV infection prevalence is widely divergent, ranging from 0% to 89%.
Nós nos interessamos na co-infecção HBV HIV pois..slide
Jeantet et al., 2002

15. Demographical and serological data of HIV positive/ HBsAg negative patients
characteristics
Years
n = 91
n = 33
Year
2006
n=43
2009
n=144
Sex
Male
Female
80 (88%)
11(12%)
21 (64%)
12(36%)
35 (81%)
8 (19%)
59 (41%)
85 (59%)
Age (years)
< 30
30-39
40-49
>49
2 (3%)
21 (29.5%)
32 (45%)
16 (22.5%)
3 (10%)
16 (51.5%)
8 (25.5%)
4 (13%)
0 (0%)
7 (16.5%)
19 (45.5%)
16 (38%)
24 (19%)
42 (33.3%)
40 (31.7%)
20 (16%)
Antiretroviral treatment
Yes No
26 (31%)
58(69%)
26 (78%)
7 (22%)
39 (90%)
4 (10%)
105 (82%)
23 (18%)
Lamivudine
33 (36%)
58 (64%)
23 (70%)
10(30%)
37 (95%)
2 (5%)
103 (80%)
25 (20%)
TDF
10 (23%0
40 (30%)
HBV markers
Anti-HBc total
87 (97%)
26 (81%)
39 (91%)
23 (16%)
Anti-HBc only
34/89 (38%)
8/30 (26%)
11/42 (26%)
4/144 (3%)
Anti-HBs/Anti-HBc
53 (59.5)
18 (60%)
28 (66.5%)
19 (13.5%)
Anti-HBs only
2/89 (2%)
1/32 (3%)
3/42 (7%)
37/144 (28%)
No HBV serological marker
3/32 (3%)
84/144 (58%)
HBV DNA Pre S or S region
05 (5.5%)
06 (18%)
06 (14%)
05 (3.3%)
Estudamos coortes hbv/hiv ao longo do tempo (slide)
HBsAg >10% ( ), 2%

16. Serological and molecular features of HBV isolates from cases of occult infection
Patient
Sex
Year
Antiretroviral treatment
LAM/TVF
Anti-HBs/Anti-HBc
HBV loads
(copies/mL)
Genotypes S
STOP CODONS
Other mutations G M
1998 Y
N/N
Pos/Pos
104
A/A1
S36 -
P36 F
2006
Y/N
106
S216
Y100C P
1999 N
S187
P51
Neg/Pos No
Y100C,*E164D
21967
2009 ND
Q101H F134L H
1997
P65
A/A2
23818
Neg/Neg
2003
102 D
T118V, A128V E
S136Y
* LAM triple mutation
HindIII
Isolados de HBV caracterizados de casos de infecção oculta: stop codon outras, algumas expressas
EcoRV
HindIII
EcoRV
Transfection of eukariotic cells (CHO or HUH7)
pre-S2 S
HindIII
EcoRV
pcDNA3 S
BIOELISA
HBsAg Colour (Biokit)

17. Expression of HBsAg containing Y100C variant frequently detected in occult HBV infection

18. Conclusions/Perspectives
Ongoing studies at IPEC-Fiocruz should confirm or not the recent decrease of HBV prevalence in the HIV infected Brazilian population, observed by us.
Drug resistance: In agreement with dr. Couto-Fernandez (member of National Network for HIV Genotyping Lab. AIDS, IOC) we had designed a doctoral thesis to study the lamivudine and tenofovir resistance mutations of both HIV and HBV (populations and subpopulations by pyrosequencing) in cases of co-infectin. Unfortunately, the collaboration did not happen. We are inviting co-infected patients from IPEC to initiate this study.
Regiã
Pol HIV

19. Conclusions/Perspectives
Studies of occult HBV infection in the sera and PBMC of HIV co-infected patients will also be carried out in France
Future studies are needed to monitor transmission of HBV isolates from cases of occult infections/drug resistance
The most appropriate therapy for a particular genotype or a mutation type may be identified by in vitro phenotyping Test: Transfection of complete genomes into human cells in the presence of drugs
The study of mutations affecting HBeAg expression in Brazilian-specific genotypes/subtypes will also be continued

20. Valorization of the project (1)
Publications
Mello, Francisco C. A. ; Martel, Nora ; Gomes, Selma A. ; Araujo, Natalia M. .Expression of Hepatitis B Virus Surface Antigen Containing Y100C Variant Frequently Detected in Occult HBV Infection. Hepatitis Research and Treatment, v. 2011, p. 1-4,
Araujo, N. M. ; Branco-Vieira M ; Silva ACM ; Pilotto JH ; Grinsztejn B ; Trepo C; Gomes, SA . Occult hepatitis B virus infection in HIV-infected patients:Evaluation of biochemical, virological and molecular parameters.. Hepatology Research, v. 38, p , 2008
Araujo NM, Waizbort R, Kay A. Hepatitis B Virus infection from an evolutionary point of view: how viral, host, and environmental factors shape genotypes and subgenotypes. Infectious, Genetics and Evolution, (under revision)
2 other papers in preparation
1 Brazilian patent deposited
1 thesis in co-tutelle Instituo Oswaldo Cruz/Université Claude Bernard Lyon 1

21. PhD student, ANRS doctoral bursary
Valorization of the project (2)
A mini-symposium organized by Selma GOMES:
“International Conferences and Debates on Relevant Aspects of Viral Hepatitis”
IOC-FIOCRUZ, Rio de Janeiro, Brazil, February 26ty 2010
Participants:
Selma GOMES, IOC-FIOCRUZ, Brazil: Introduction
Anna KRAMVIS, Witwatersrand University, Johannesburg, South Africa:
Phylogeny of Hepatitis B Virus
Stephen Locarnini, Victoria Infectious Diseases Reference Laboratory,
Melbourne, Australia: Drug resistance of Hepatitis B Virus
Christoph COMBET, IBCP, Lyon, France: Hepatitis C virus genomic variability
Alan Kay, INSERM U871, Lyon, France:Cell-culture models for HBV infectivityulture models for HBV infectivity
Exchanges
Rio de Janeiro  Lyon
Lyon  Rio de Janeiro
Natalia
ARAUJO
Caroline
SOARES
Nora MARTEL
PhD student, ANRS doctoral bursary
Joint thesis IOC/UCBL1

22. Research team/ Acknowledgment
Brazilian team
Researcher
Dr. Natalia M. Araujo
PHD students
Kelly C. Pereira
French team
Researchers/clinicians
Dr Isabelle Chemin
Pr. Christian Trepo
Dr. Laurent Cotte
Dr. Sophie Allain (Limoges
PHD student
Nora Martel
Collaborators (cohort of patients)
IPEC/Fiocruz (Dr.Beatriz Grinsztejn, Dr.Juçara Arabe
Other Financial Support
Brazil: CNPq/ France/Brazil: INSERM/FIOCRUZ