- Email: [email protected]
WITHDRAWN: T-cell responses to the hepatitis B core antigen identified occult hepatitis B infection genotype F with HBV DNA fluctuation between undetectable and high viral loads
Accepted Manuscript Title: T-cell responses to the hepatitis B core antigen identified occult hepatitis B infection genotype F with HBV DNA fluctuation between undetectable and high viral loads Author: Araujo Patricia Flavia Latini Juliana Cardoso Ricardo Sobhie Diaz PII: DOI: Reference:
S2214-2509(16)00003-2 http://dx.doi.org/doi:10.1016/j.idcr.2016.01.002 IDCR 86
To appear in: Received date: Revised date: Accepted date:
21-7-2015 19-8-2015 9-1-2016
Please cite this article as: Patricia A, Latini F, Cardoso J, Diaz RS, T-cell responses to the hepatitis B core antigen identified occult hepatitis B infection genotype F with HBV DNA fluctuation between undetectable and high viral loads., IDCR (2016), http://dx.doi.org/10.1016/j.idcr.2016.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
T-cell responses to the hepatitis B core antigen identified occult hepatitis B infection genotype F with HBV DNA fluctuation between undetectable and high viral loads.
Authors and Institutions: Patricia, Araujo1,3; Flavia Latini1; Juliana Cardoso and Ricardo Sobhie
ip t
Diaz3.
1.Colsan - Associação Beneficente de Coleta de Sangue, São Paulo, SP, Brazil.
cr
2.Histocompatibility and Criopreservation Laboratory; Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
an
us
3.Retrovirology Laboratory; Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil
ed
M
No conflict of interest
Corresponding Author: PatríciaAraújo
ce pt
Address: Avenida Indianópolis, 1260, Indianópolis, São Paulo, SP, phone 55-1150556588 São
Ac
Paulo, SP, Brazil. E-mail: [email protected]
Page 1 of 5
Abstract A 33-year-old male, first time blood donor with a past history of HBV vaccination and no declared risk factors for HBV infection, was found with an absence of reactivity to
ip t
HBV markers, HBV DNA fluctuation, and a strong and unremitting host cellular immune response to HBV detected over time. HBV genome sequencing identified a
cr
genotype F strain, subgenotype F2. This is the first case report that associated
an
us
subgenotype F2 with occult HBV infection in Brazilian blood donors.
M
Case Report Between October 1st 2010 and December 31st 2013 a total of 480,623blood donations from city of São Paulo, Brazil have been serologically screened for HBV infection in
ed
the Colsan Blood Center. In February 2011, a 33-year old male first time blood donor born in the city of São Paulo and asymptomatic, previously vaccinated against hepatitis B with no
ce pt
declared risk factors for HBV infection, including traveling to Amazon region of Brazil, presented non-reactivity to HBsAg, Anti-HBc and anti-HBs in serological screening. The donation sample of this blood donor was submitted to anHBV related research as part of a health blood donor control group, which included the investigation of HBcAg-T cell response
Ac
by lymphoproliferation, hepatitis B serological markers (anti-HBs, anti-HBc, anti-HBc IgM, HBeAg and anti-HBe), HBV DNA (Cobas HBV Monitor Assay, Roche Molecular System, Branchburg, NJ, USA,and hemi-nested PCR in the basic core promoter/precore region (BCP/PC) (Candotti et al.,2006), killer immunoglobulin like receptors (KIR) and human leukocyte antigen (HLA) genotyping using a Luminex Multi-analyte profiling system (One Lambda, Inc., Canoga Park, CA) with the LABType SSO OneLambda typing kit (One Lambda, Inc., Canoga Park, CA), Ex vivo NK cell activity assay, and Enzyme-linked immunospot assay for INF- (ELIspot-INF-) in PBMC (6).
Page 2 of 5
Results from the February 2011 donation sample revealed a confirmed undetectable HBV DNA, negative hepatitis B markers, and positive HBcAg-Tcell response (stimulation index=60,8), ELIspot-INF- (435ISCs/106 PBMCs) and NK cell activity (>80% lyses). The blood unit donated by this blood donor was therefore eliminated and classified as high-risk for
ip t
HBV infection. New sample was collected in August 2011 (about 6 months after the first donation), and
cr
a first positive test for HBV DNA was detectedrevealing a viral load 3.5x 108copies/mL, whereas all hepatitis B markers were still negative. Further, an high HBcAg-Tcell response
us
(stimulation index=56.1) was determined together with NK cells activity (>80% of lyses) and
an
positive ELIspot-INF-(1.112ISCs/106 PBMCs).In this case a specific pattern of KIR and HLAC:KIR2DL3 and HLAC1 unitalize homozygosity was identified.HBV genome sequence [1] identified a genotype F strain, subgenotype F2classified according to reference sequences from
M
GenBank. The entire genome was sequenced and a mutation in the core region leading to a
truncated or abnormal capsid protein was detected, which conceivably could affect
ed
viral/antibody production as observed in this case (G1764A) (1).No evidence of HBsAg escape
ce pt
mutants that could explain the absence of reactivity to HBsAg in this case. Phylogenetically HBV has been 9 genotypes, A–I, and a reputed 10th genotype ‘J’, isolated from a single individual. Genotypes A–D, F, H, and I are classified further into at least 35 subgenotypes. HBV genotypes/subgenotypes and genetic variability of HBV are useful in
Ac
epidemiological and transmission studies, tracing human migrations, and in predicting the risk for the development of severe liver disease and response to antiviral therapy. The genotypes, and certain subgenotypes, have distinct geographical distributions and are important in both the clinical manifestation of infection and response to antiviral therapy (7). The genotype identified in this report case, genotype F, is usually found in the North region of Brazil (6).being rare in other parts of this country (2) and at our best knowledge, we do not find any report about occult HBV infection(OBI) among subgenotype F2 in last line Brazilian blood banks.
Page 3 of 5
Following the first demonstration of HBV in August, 2011; the blood donor was further monitored in February 2012, June 2012 November 2012, March 2013, September 2013 and March 2014.In all donationssubsequent to August 2011a non-reactivityfor hepatitis B markers and fluctuations in HBV-DNA levels have been observed. Immunologic parameters remained without significance alterations.
ip t
We detected HBV DNA only on February 2012 and November 2012, with anextremely
high viral load of4.1x 108copies/mL and 3.7x 108copies/mL of plasma respectively. At the
cr
monitored period, NK cells activity (<70% median of lyses), HBcAg-specific T cell responses
us
(SIM=59.1) and ELIspot-INF- (median=1.141 ISCs/106 PBMCs) were positive in all different time points.The patient was completely asymptomatic with normal ALT/AST levels in all study
an
period, but unfortunately, we did not have access toother laboratory analysis or any other clinicalfollow-up of this patient. Nonetheless, this specific patient would be suitable as a blood
M
donor at all investigated time points using the regular screening procedures because HBV NAT is not mandatory at last line Brazilian blood banks.
ed
Discussion in this particular case, HBcAg-T cell response was fundamental to identify this particular occult HBV infection in its first time donation in the absence of HBV DNA and
ce pt
hepatitis B markers. The reasons for the HBV DNA fluctuation levels in the absence of hepatitis B serological markers in OBI remain unknown. However, it can be speculated that both host and viral factors are important in intermittently controlling viral replication and also controlling
Ac
the disease progression (3-6). It has been previously showed that the interaction between KIR genes and HLA is important in determining antiviral immunity and contributes to the resolution or persistence of HBV infection. Interestingly, the specific pattern of KIR and HLA-C: KIR2DL3 and HLAC1 unitalize homozygosity found in this reported case have been previously associated with viral clearance (4). The role of NK cells in viral clearance during acute HBV infection is also supported by previous reports showing that early high IFN- production by NK cells may contribute to the initial control of the infection and allow the timely development of an adaptive immune
Page 4 of 5
response (4).In this reported case, despite the higher specific T-cell response, INF- production,activity of NK cells activity and KIR/HLA interaction associated with viral clearance,the viral fluctuation was observed in study period due to probably serious HBV humoral response alteration and the complex interaction between host and virus.
ip t
.
cr
References
an
us
1.Candotti D, Opare-Sem O, Rezvan H,Sarkodie F and Allain JP.Molecular and serological characterization of hepatitis B virus in deferred Ghanaian blood donors with and without elevated alanine aminotransferase.JViral Hep2006;13:715–724.
M
2. Mónica V, Alvarado-Mora L, Botelho-Lima L, Santana RA, Sitnik R, Ferreira PA,Mello AF, Mangueira CP, Carrilho FJ, Pinho JRR.Distribution of hepatitis B virus subgenotype F2a in São Paulo, Brazil.BMC Res Notes. 2013;6: 423.
ed
3.Chang J,Block TM,Guo JT. The innate immune response to hepatitis B virus infection: implications for pathogenesis and therapy.Antiviral Res.2012;96(3):405-13. 4. Araujo PR, Goncalves G, Latini F, Ferreira O, Porto LC, Barreto JA, Girao MJC and Diaz RS..KIR and a specific HLA-C gene are associated with susceptibility and resistance to hepatitis B virus infection in a Brazilian population.CellMolImmunol2014:1–4
ce pt
5. Li S,Gowans EJ, Chougnet C, Plebanski M andDittmer U. Natural Regulatory T Cells and Persistent.Viral Infection J. Virol.2008;82(1):21-30. 6. Kiesslich D, Crispim MA, Santos C, Ferreira FL, Fraiji NA, Komninakis SV, Diaz RS. Influence of hepatitis B virus genotype in hepatitis B virus/hepatitis delta virus dual infection. JInfectDis. 2009 1;199(11):1608-11.
Ac
7. Anna Kramviss. Genotypes and genetic variability of HBV. Intervirology. 2014 57:141150
Page 5 of 5
S2214-2509(16)00003-2 http://dx.doi.org/doi:10.1016/j.idcr.2016.01.002 IDCR 86
To appear in: Received date: Revised date: Accepted date:
21-7-2015 19-8-2015 9-1-2016
Please cite this article as: Patricia A, Latini F, Cardoso J, Diaz RS, T-cell responses to the hepatitis B core antigen identified occult hepatitis B infection genotype F with HBV DNA fluctuation between undetectable and high viral loads., IDCR (2016), http://dx.doi.org/10.1016/j.idcr.2016.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
T-cell responses to the hepatitis B core antigen identified occult hepatitis B infection genotype F with HBV DNA fluctuation between undetectable and high viral loads.
Authors and Institutions: Patricia, Araujo1,3; Flavia Latini1; Juliana Cardoso and Ricardo Sobhie
ip t
Diaz3.
1.Colsan - Associação Beneficente de Coleta de Sangue, São Paulo, SP, Brazil.
cr
2.Histocompatibility and Criopreservation Laboratory; Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
an
us
3.Retrovirology Laboratory; Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil
ed
M
No conflict of interest
Corresponding Author: PatríciaAraújo
ce pt
Address: Avenida Indianópolis, 1260, Indianópolis, São Paulo, SP, phone 55-1150556588 São
Ac
Paulo, SP, Brazil. E-mail: [email protected]
Page 1 of 5
Abstract A 33-year-old male, first time blood donor with a past history of HBV vaccination and no declared risk factors for HBV infection, was found with an absence of reactivity to
ip t
HBV markers, HBV DNA fluctuation, and a strong and unremitting host cellular immune response to HBV detected over time. HBV genome sequencing identified a
cr
genotype F strain, subgenotype F2. This is the first case report that associated
an
us
subgenotype F2 with occult HBV infection in Brazilian blood donors.
M
Case Report Between October 1st 2010 and December 31st 2013 a total of 480,623blood donations from city of São Paulo, Brazil have been serologically screened for HBV infection in
ed
the Colsan Blood Center. In February 2011, a 33-year old male first time blood donor born in the city of São Paulo and asymptomatic, previously vaccinated against hepatitis B with no
ce pt
declared risk factors for HBV infection, including traveling to Amazon region of Brazil, presented non-reactivity to HBsAg, Anti-HBc and anti-HBs in serological screening. The donation sample of this blood donor was submitted to anHBV related research as part of a health blood donor control group, which included the investigation of HBcAg-T cell response
Ac
by lymphoproliferation, hepatitis B serological markers (anti-HBs, anti-HBc, anti-HBc IgM, HBeAg and anti-HBe), HBV DNA (Cobas HBV Monitor Assay, Roche Molecular System, Branchburg, NJ, USA,and hemi-nested PCR in the basic core promoter/precore region (BCP/PC) (Candotti et al.,2006), killer immunoglobulin like receptors (KIR) and human leukocyte antigen (HLA) genotyping using a Luminex Multi-analyte profiling system (One Lambda, Inc., Canoga Park, CA) with the LABType SSO OneLambda typing kit (One Lambda, Inc., Canoga Park, CA), Ex vivo NK cell activity assay, and Enzyme-linked immunospot assay for INF- (ELIspot-INF-) in PBMC (6).
Page 2 of 5
Results from the February 2011 donation sample revealed a confirmed undetectable HBV DNA, negative hepatitis B markers, and positive HBcAg-Tcell response (stimulation index=60,8), ELIspot-INF- (435ISCs/106 PBMCs) and NK cell activity (>80% lyses). The blood unit donated by this blood donor was therefore eliminated and classified as high-risk for
ip t
HBV infection. New sample was collected in August 2011 (about 6 months after the first donation), and
cr
a first positive test for HBV DNA was detectedrevealing a viral load 3.5x 108copies/mL, whereas all hepatitis B markers were still negative. Further, an high HBcAg-Tcell response
us
(stimulation index=56.1) was determined together with NK cells activity (>80% of lyses) and
an
positive ELIspot-INF-(1.112ISCs/106 PBMCs).In this case a specific pattern of KIR and HLAC:KIR2DL3 and HLAC1 unitalize homozygosity was identified.HBV genome sequence [1] identified a genotype F strain, subgenotype F2classified according to reference sequences from
M
GenBank. The entire genome was sequenced and a mutation in the core region leading to a
truncated or abnormal capsid protein was detected, which conceivably could affect
ed
viral/antibody production as observed in this case (G1764A) (1).No evidence of HBsAg escape
ce pt
mutants that could explain the absence of reactivity to HBsAg in this case. Phylogenetically HBV has been 9 genotypes, A–I, and a reputed 10th genotype ‘J’, isolated from a single individual. Genotypes A–D, F, H, and I are classified further into at least 35 subgenotypes. HBV genotypes/subgenotypes and genetic variability of HBV are useful in
Ac
epidemiological and transmission studies, tracing human migrations, and in predicting the risk for the development of severe liver disease and response to antiviral therapy. The genotypes, and certain subgenotypes, have distinct geographical distributions and are important in both the clinical manifestation of infection and response to antiviral therapy (7). The genotype identified in this report case, genotype F, is usually found in the North region of Brazil (6).being rare in other parts of this country (2) and at our best knowledge, we do not find any report about occult HBV infection(OBI) among subgenotype F2 in last line Brazilian blood banks.
Page 3 of 5
Following the first demonstration of HBV in August, 2011; the blood donor was further monitored in February 2012, June 2012 November 2012, March 2013, September 2013 and March 2014.In all donationssubsequent to August 2011a non-reactivityfor hepatitis B markers and fluctuations in HBV-DNA levels have been observed. Immunologic parameters remained without significance alterations.
ip t
We detected HBV DNA only on February 2012 and November 2012, with anextremely
high viral load of4.1x 108copies/mL and 3.7x 108copies/mL of plasma respectively. At the
cr
monitored period, NK cells activity (<70% median of lyses), HBcAg-specific T cell responses
us
(SIM=59.1) and ELIspot-INF- (median=1.141 ISCs/106 PBMCs) were positive in all different time points.The patient was completely asymptomatic with normal ALT/AST levels in all study
an
period, but unfortunately, we did not have access toother laboratory analysis or any other clinicalfollow-up of this patient. Nonetheless, this specific patient would be suitable as a blood
M
donor at all investigated time points using the regular screening procedures because HBV NAT is not mandatory at last line Brazilian blood banks.
ed
Discussion in this particular case, HBcAg-T cell response was fundamental to identify this particular occult HBV infection in its first time donation in the absence of HBV DNA and
ce pt
hepatitis B markers. The reasons for the HBV DNA fluctuation levels in the absence of hepatitis B serological markers in OBI remain unknown. However, it can be speculated that both host and viral factors are important in intermittently controlling viral replication and also controlling
Ac
the disease progression (3-6). It has been previously showed that the interaction between KIR genes and HLA is important in determining antiviral immunity and contributes to the resolution or persistence of HBV infection. Interestingly, the specific pattern of KIR and HLA-C: KIR2DL3 and HLAC1 unitalize homozygosity found in this reported case have been previously associated with viral clearance (4). The role of NK cells in viral clearance during acute HBV infection is also supported by previous reports showing that early high IFN- production by NK cells may contribute to the initial control of the infection and allow the timely development of an adaptive immune
Page 4 of 5
response (4).In this reported case, despite the higher specific T-cell response, INF- production,activity of NK cells activity and KIR/HLA interaction associated with viral clearance,the viral fluctuation was observed in study period due to probably serious HBV humoral response alteration and the complex interaction between host and virus.
ip t
.
cr
References
an
us
1.Candotti D, Opare-Sem O, Rezvan H,Sarkodie F and Allain JP.Molecular and serological characterization of hepatitis B virus in deferred Ghanaian blood donors with and without elevated alanine aminotransferase.JViral Hep2006;13:715–724.
M
2. Mónica V, Alvarado-Mora L, Botelho-Lima L, Santana RA, Sitnik R, Ferreira PA,Mello AF, Mangueira CP, Carrilho FJ, Pinho JRR.Distribution of hepatitis B virus subgenotype F2a in São Paulo, Brazil.BMC Res Notes. 2013;6: 423.
ed
3.Chang J,Block TM,Guo JT. The innate immune response to hepatitis B virus infection: implications for pathogenesis and therapy.Antiviral Res.2012;96(3):405-13. 4. Araujo PR, Goncalves G, Latini F, Ferreira O, Porto LC, Barreto JA, Girao MJC and Diaz RS..KIR and a specific HLA-C gene are associated with susceptibility and resistance to hepatitis B virus infection in a Brazilian population.CellMolImmunol2014:1–4
ce pt
5. Li S,Gowans EJ, Chougnet C, Plebanski M andDittmer U. Natural Regulatory T Cells and Persistent.Viral Infection J. Virol.2008;82(1):21-30. 6. Kiesslich D, Crispim MA, Santos C, Ferreira FL, Fraiji NA, Komninakis SV, Diaz RS. Influence of hepatitis B virus genotype in hepatitis B virus/hepatitis delta virus dual infection. JInfectDis. 2009 1;199(11):1608-11.
Ac
7. Anna Kramviss. Genotypes and genetic variability of HBV. Intervirology. 2014 57:141150
Page 5 of 5