Hepatitis C virus (HCV)-specific memory B-cell responses in transiently and chronically infected HIV positive individuals

Journal of Clinical Virology 59 (2014) 218–222

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Hepatitis C virus (HCV)-specific memory B-cell responses in transiently and chronically infected HIV positive individuals Sven Reiche a,1 , Claudia Nestler a , Michael Sieg a , Katharina Schulz a , Christiane Cordes b , Ivanka Krznaric c , Christian Jassoy a,∗ a

Institute of Virology, Faculty of Medicine, University of Leipzig, 04105 Leipzig, Germany Medical Practice Dr. Cordes, Berlin, Germany c Medical Center for Infectious Diseases, Berlin, Germany b

a r t i c l e

i n f o

Article history: Received 1 August 2013 Received in revised form 3 December 2013 Accepted 17 January 2014 Keywords: Hepatitis C virus HIV Memory B-lymphocytes Pathogenesis

a b s t r a c t Background: Antibody responses to hepatitis C virus (HCV) occur delayed and overly decline after viral clearance indicating that the B-cell response to HCV is abnormal. Virus-specific memory B-cells have recently been found in infected individuals, but the viral exposure requirements for the generation of these cells is unknown. Objectives: The primary goal of this study was to quantify and compare the HCV-specific memory B-cell response between chronic and resolved HCV-infected individuals. A secondary goal was to examine if HIV-specific memory B-cell responses are maintained during HCV co-infection. Study design: HCV core protein- and HIV-specific memory B-cell responses were examined in HIV/HCVinfected individuals treated 4–30 weeks after HCV diagnosis. Memory B-cell frequencies were compared between chronically and transiently infected individuals. Results: Chronically infected individuals had vigorous HCV-specific memory B-cell responses and antibodies, whereas subjects with transient viremia showed low or undetectable virus-specific B-cell responses. In addition, chronically HIV/HCV-infected subjects had robust HIV-specific memory B-cell responses. Conclusions: Whereas chronic HCV infection induces virus-specific antibodies and memory B-cells, transient infection in individuals with sustained viral response to therapy does not stimulate a durable HCV-specific B-cell response indicating that the formation of long-lived virus-specific B-cells is suppressed in the early phase of infection. This may contribute to the inability to spontaneously clear HCV infection. © 2014 Elsevier B.V. All rights reserved.

1. Background The role of B-cells in protection from chronic HCV infection is currently unclear [1]. Spontaneous viral clearance occasionally occurs without seroconversion and has been observed in agammaglobulinemic twins indicating that virus-specific antibodies are not required for immune protection [2,3]. On the other hand, it was shown in a chimpanzee model that neutralizing antibodies can be protective [4,5]. Antibodies to HCV develop slowly with a mean time to seroconversion of 6 weeks indicating that the development of virus-specific plasma cells is delayed. In addition, antibodies

Abbreviations: HCV, hepatitis C virus; HIV, human immunodeficiency virus; SVR, sustained viral response; NP, nucleoprotein; Ttx, tetanus toxoid. ∗ Corresponding author at: Institute of Virology, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany. Tel.: +49 341 9714314; fax: +49 341 9714309. E-mail address: [email protected] (C. Jassoy). 1 S.R. and C.N. contributed equally to the study. 1386-6532/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2014.01.023

decrease relatively rapidly after spontaneous viral clearance and therapeutic eradication suggesting that the immune response is not long-lived [6–12]. This is different from antibody responses against other viral infections that decline with half-lives of several decades and may indicate that HCV affects the generation of virus-specific long-lived B-cells [13,14]. HCV-specific memory B-cells have recently been demonstrated in HCV-monoinfected individuals [15], but the requirements in terms of duration of infection for the development of these cells have not yet been studied. It is estimated that 10 million persons infected with hepatitis C virus (HCV) are co-infected with the human immunodeficiency virus (HIV) worldwide [16]. Epidemiologic data show that the prevalence of HCV infection is particularly increasing in HIV-infected men who have sex with men (MSM) and routine screening of acute HCV infection is warranted [16,17]. We have recently shown that HIV-infected individuals with CD4+ T-cell numbers above 350 cells/␮l have vigorous HIV-specific memory B-cell responses, whereas memory B-cell numbers were decreased

S. Reiche et al. / Journal of Clinical Virology 59 (2014) 218–222

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in subjects with helper T-cell frequencies below 200 cells/␮l indicating that the loss of helper T-cells affects the ability to sustain HIV-specific memory B-cells [18]. While previous studies showed that HCV has little effect on HIV infection, it has not yet been reported whether HIV-infected individuals maintain virus-specific responses after secondary infection with HCV [17]. As we were regularly testing HIV-infected individuals at risk for signs of viral hepatitis, acute HCV infection was recognized early in these individuals and patients were treated with interferon and ribavirin. This afforded the opportunity to study the memory B-cell response to HIV and HCV in subjects with transient HCV viremia and in non-responders with chronic HCV infection

Wernigerode GmbH). CD19-mediated selection of the cells had no negative effect on subsequent measurements (data not shown). PBMCs depleted of B-cells were treated with mitomycin C (50 mg/l, Merck KgaA) and used as feeder cells. B-cells (2 × 105 ) and feeder cells (1.8 × 106 ) were cultured for 6 days at 37 ◦ C with a mixture of mitogenic agents including pokeweed mitogen (PWM, Sigma–Aldrich, Inc.), Staphylococcus aureus lysate (Sigma–Aldrich, Inc.), interleukin (IL)-2 (Proleukin, Novartis AG), IL-10 (Hiss Diagnostics GmbH) and phosphorothioated CpG ODN-2006 (Metabion GmbH) to induce differentiation to plasma cells.

2. Objective

Antibody secretion was examined by ELISpot using 96-well Multiscreen HA plates (Millipore, Inc.). Total numbers of antibody secreting cells were determined with plates coated with goat anti-human Ig recognizing IgG F(ab )2 (Dianova GmbH) at 4 ◦ C over night. Antigen-specific responses were examined with plates coated with HCV core protein (2 ␮g/well), Gag/p24 (2 ␮g/well), influenza virus NP (2 ␮g/well) or Ttx (1 ␮g/well) in duplicates. Wells coated with MBP (1 ␮g/well) were used as controls. After washing and blocking of the wells with RPMI-1640 medium/10% FCS for 2 h at 37 ◦ C, 100 (Ig-coated wells), 100,000 (NP and Ttxcoated wells) or 200,000 (Gag/p24, HCV core and MBP-coated wells) cultured cells were added and the plates incubated for 20 h at 37 ◦ C. Plates were washed and alkaline phosphatase-conjugated goat anti-human Ig recognizing IgG (Fc-fragment, Dianova GmbH) was added for 2 h. The plates were developed using the AP Conjugate Substrate Kit (Bio-Rad Laboratories, Inc.). Spots were counted using an AID ELISpot 04 plate reader (Autoimmun Diagnostika GmbH). Spots in wells with control proteins were subtracted from the number of spots in wells with antigen. The frequency of antigen-specific cells was calculated as the percentage of total antibody-secreting cells according to the formula:

The primary goal of this study was to find HCV-specific memory B-cells in HIV/HCV-coinfected individuals and to compare the virus-specific memory B-cell response in chronic disease with that after transient infection. In addition, we were also interested to examine if HIV-specific memory B-cells were maintained during HCV co-infection. 3. Materials and methods 3.1. Study participants Thirteen subjects were diagnosed with HIV 1–24 years (mean 8.2 years) prior to HCV. Two of the participants presumably had a co-infection already at the beginning of the study. HCV infection was diagnosed by commercial HCV antibody ELISA and RT-PCR. HIV and HCV RNA were quantified by real-time RT-PCR (Abbott). All subjects were males who have sex with males. The subjects were treated with interferon-␣ and ribavirin between 5 and 30 weeks (average 14 weeks) after diagnosis of HCV infection. Immune responses were tested 35.7 months (range 12–81 months) after diagnosis of the infection. Informed consent was obtained from the subjects and the study was approved by the Ethics Committee at the Faculty of Medicine of the University of Leipzig.

3.4. B-cell ELISpot

%Ag-specific spots =

Ag spots − control spots 100 · × 100 lg spots 100, 000 or 200, 000

3.2. Antigens

3.5. Antibody ELISA

Recombinant HCV core protein (amino acids 1–191) was prepared from viral RNA of a HCV subtype 1b positive clinical sample (strain 09/08099). The RNA was reverse transcribed and the cDNA cloned in a modified pMALc2x expression plasmid (New England Biolabs, Inc.). The protein was expressed as a maltose binding protein fusion protein in Escherichia coli and purified with an amylose resin solution. HIV Gag/p24 and influenza virus (H3N2) nucleoprotein (NP) were prepared similarly, as recently described [19]. Tetanus toxoid was obtained from Novartis-Behring, Marburg, Germany, and GlaxoSmithKline, Rixensart, Belgium, provided recombinant HIV-1W61D gp120 expressed in Chinese hamster ovary cells.

96-Well microtiter plates (Greiner Bio-One GmbH) were coated overnight with recombinant protein (0.05–0.1 ␮g/well) in coating buffer (0.2 M NaHCO3 , pH 9.6). The plates were washed with phosphate-buffered saline/0.05%Tween-20 (PBST) and blocked with 5% milk powder in PBST. Sera were fivefold serially diluted with PBST/milk powder, added to the wells and incubated for 2 h at 37 ◦ C. HRP-conjugated rabbit anti-human IgG (Dako GmbH, diluted 1:5000) was added for 1.5 h. Plates were incubated for 30 min at room temperature with developing solution. The reaction was stopped with H2 SO4 and the OD at 450 nm was determined. Measurements were done in duplicates. Midpoint titers were calculated. Midpoint titers were defined as the reciprocal value of the serum dilution at half maximal OD after background subtraction [20]. Pooled serum from HCV-infected individuals was used as reference and tested in each series of experiments to adjust the half maximal OD values. A fictitious antibody titer of 10 was taken for those sera that did not reach half-maximum OD at a dilution of 1:50.

3.3. B-cell isolation and activation Peripheral blood mononuclear cells (PBMCs) were prepared from heparinized blood by density gradient centrifugation using a ficoll cushion (Histopaque, Sigma–Aldrich, Inc.). B-cells were isolated with immunomagnetic anti-CD19 beads (Dynal® Pan CD19 kit, Invitrogen, Inc.). Cells were resuspended in RPMI-1640 medium (Invitrogen, Inc.) supplemented with 10% fetal calf serum (Biochrom AG), 1% non essential amino acids, 1% sodium pyruvate, 1% l-glutamine (Invitrogen, Inc.), penicillin (100 IU/l, Jenapharm GmbH & Co. KG) and streptomycin sulfate (100 mg/l, Pharma

3.6. Statistical analyses Statistical analyses were performed using SPSS software. The Mann–Whitney test was used to do pairwise comparisons of the groups.

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Table 1

HCV infection

Subject no.

CD4 T-cell count (c/␮l)a

Start of IFN/RBV treatment after diagnosis (days)

B-cell testing after diagnosis (months)

Chronic

7 12 22 24 28 34

373 376 399 194 400 379

130 60 66 213 188 60

31 13 12 45 87 60

353.5

130.3b

41.3

665 332 704 462 389 175 600 553 694

119 74 50 60 55 139 176 38 107

Mean SVR

3 5 8 18 19 20 23 26 29

a b c

91.3b

508

Mean

Minimum HCV exposure (weeks)

Maximum RNA concentration (IU/ml)

HCV core-specific antibody titer

HCV core-specific memory B-cells (%)

– – – – – –

– – – – – –

29,765 97,669 10 52,702 95,445 15,456

0.023 0.405 <0.001 0.109 0.124 0.006

45 21 29 50 37 14 27 21 44

21 15 7 24 6 24 31 6 14

345,000 12,800,000 482,000 832,000 1,170,000 12,000,000 768,000 15,300,000 974,000

<10 <10 <10 <10 <10 <10 <10 <10 <10

<0.001 0.005 <0.001 0.002 <0.001 <0.001 <0.001 <0.001 0.008

32

16

1,170,000c

CD4+ T-cell count at diagnosis of HCV infection or up to three months before. The mean start of therapy after diagnosis in subjects with chronic infection and with SVR was not statistically significant (Mann–Whitney test, p = 0.328). Median.

4. Results Study participants were assigned to the group of chronically infected subjects that did not fully respond to antiviral therapy or the group with a sustained viral response (SVR). Groups were similar with respect to age (mean 34.7 versus 40.4 years, p = 0.087), sex (all males), and CD4+ T-cell count around HCV diagnosis (354 versus 508, p = 0.099). B-cell responses in the chronically infected group were tested 41 months (range 12–81 months) after diagnosis of HCV infection. Subjects in the SVR group had HCV viremia for an average minimum of 16 weeks (range 6–31 weeks). During this period, between 2 and 5 quantitative RNA measurements were positive. The true time of viral exposure was longer since only the time between the first and last positive HCV RNA test was used for the analysis. Most individuals had high viral loads. The maximum RNA concentrations were between 4.82 × 105 and 15.3 × 106 IU/ml. Subjects with SVR were anti-HCV antibody positive by commercial ELISA at the time of diagnosis. HCV core-specific antibody and memory B-cell responses were tested 32 months (range 14–50 months) after diagnosis (Table 1). All individuals but one in the chronically infected group showed a HCV-specific memory B-cell and antibody response. In contrast, only three of the nine subjects with SVR had HCV-specific memory B-cells and none of these had measurable levels of virus core-specific antibodies. These differences were statistically significant and indicate that transient infection with high

5. Discussion The antibody response to HCV has previously been examined in numerous studies. It was shown that seroconversion takes several weeks and antibodies frequently decrease after spontaneous viral clearance and therapeutic eradication [6–12]. However, little is known about virus-specific memory B-cells. Low frequencies of virus-specific B-cells have recently been found in chronic infection [15] but the consequences of transient infection for the generation of these cells have not yet been determined. To examine the development of memory B-cells, we studied groups of HCV-infected patients with transient and chronic infection. We demonstrated that the majority of chronically infected individuals had vigorous HCV core-specific memory B-cell responses one to six years after 1000000

p = 0.018

Antibody titre

1

% Memory B-cells

viral loads did not induce durable B-cell responses (Fig. 1 and Table 1). The majority of the chronically HIV/HCV-infected subjects had robust antibody and memory B-cell responses to HIV Gag and Env/gp120 similar to that previously observed in HIV-infected individuals under therapy [18]. Thus, there was no evidence that HCV co-infection reduced the HIV-specific memory B-cell response. Side by side comparison of HIV monoinfected and HIV/HCV co-infected individuals is required to answer this question conclusively. Influenza NP and Ttx-specific responses were robust and comparable between the groups (Fig. 2).

0.1

0.01

p = 0.005

100000 10000 1000 100

0.001

10 SVR

chronic

HCV core

SVR

chronic

HCV core

Fig. 1. HCV-specific memory B-cell and antibody responses in chronic HIV/HCV infection and in individuals with SVR. Memory B-cell frequencies and antibody titers to HCV core protein in chronically (chronic) and transiently (SVR) HIV/HCV-infected individuals. Bars show median values.

S. Reiche et al. / Journal of Clinical Virology 59 (2014) 218–222

Antibody titre

% Memory B-cells

1000000

p = 0.388

1

0.1

0.01

p = 0.955

100000 10000 1000 100 10

0.001

HIV p24

HIV p24

Antibody titre

% Memory B-cells

p = 0.529

1000000

p = 0.864

1

0.1

0.01

100000 10000 1000 100 10

0.001

HIV gp120

HIV gp120

p = 0.864

1000000

p = 0.224 Antibody titre

1

SVR

chronic

SVR

chronic

% Memory B-cells

SVR

chronic

SVR

chronic

0.1

0.01

100000 10000 1000 100 10

0.001

Influenza NP

Influenza NP

1000000

p = 0.776 Antibody titre

10

SVR

chronic

SVR

chronic

% Memory B-cells

221

1 0.1 0.01

p = 0.181

100000 10000 1000 100 10

0.001

SVR

chronic Ttx

SVR

chronic Ttx

Fig. 2. HIV-, influenza NP and tetanus toxoid-specific memory B-cell and antibody responses. HIV Gag protein p24, HIV Env/gp120, influenza nucleoprotein (NP) and tetanus toxoid (Ttx)-specific memory B-cell and antibody responses in chronically (chronic) and transiently (SVR) HIV/HCV-infected individuals. Bars show median values.

infection, whereas virus-specific memory B-cell responses were rarely detected in individuals transiently infected for several weeks with maximum viral loads between 345,000 and 15,300,000 IU/ml. This adds to previous studies that showed that HCV-specific antibodies are present in chronic infection and decreased after viral clearance [6–12] and suggests that the development of stable HCVspecific memory B-cells requires extended exposure to the virus or is inhibited during early infection. As the time point of infection is unknown in most cases of HCV infection, we studied HIV-infected individuals that were at risk of HCV infection and therefore regularly screened for HCV. In these patients, the time point of HCV infection was diagnosed early and treatment was started in the following weeks. The number of subjects was relatively small due to the limited number of individuals available that fulfilled the inclusion criteria. However, the group size was comparable with that of other recent reports with rare patient groups. The study by Netski et al. retrospectively examined sets of sera from 12 individuals with acute HCV infection and found that the humoral immune response to HCV was delayed and of relatively low titer [7]. Our study adds to this observation and indicates that transient infection does not stimulate a lasting antibody

and memory B-cell response. Burbelo and colleagues prospectively examined HCV antibody titers in 29 chronically infected patients. Their study showed significant decreases of antibodies in a group of 9 individuals with SVR but not in groups of subjects with relapse or non-responders [6]. It would be interesting to examine if maintenance of the virus-specific memory B-cell response similarly depends on viral persistence once it has been established. While neutralizing antibodies are directed against the glycoproteins, the HCV core antigen was chosen as a marker for memory B-cells against HCV based on previous reports that core-specific antibody responses are frequently observed, more vigorous than antibody responses to the viral glycoproteins and more stable after viral clearance than antibody responses to other HCV proteins [7,9,10,21]. A critical question is why individuals with several weeks of HCV viremia did not develop robust and lasting antibody and memory B-cell responses. The weak B-cell response after transient HCV infection contrasts with that after acute viral infections such as measles, mumps and rubella. These infections have short viremic phases of three to four weeks [22–24] but induce vigorous antibody and memory B-cell responses with half-lives of many years to

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decades [14]. Similarly, smallpox vaccination induces a persistent antibody and memory B-cell response [13]. As HCV infection in the subjects with SVR lasted for an average minimum of 16 weeks and viral loads were as high as 15.3 × 106 IU/ml, it is unlikely that antigen exposure time was too short and the amount of HCV antigen was too low to generate a lasting B-cell response. Instead, the lack of HCV-specific antibodies and the low frequency with which memory B-cells were detected 1–6 years after acute infection suggest that HCV causes a state of immune tolerance. As long-lived plasma and memory B-cells are generated mainly in the germinal center reaction in secondary follicles [25] the lack of HCV-specific antibodies and memory B-cells suggests that the infection interferes with the formation of virus-specific plasma and memory B-cells before or during the germinal center reaction in the lymphatic tissue. Funding The work was supported by a Young Researcher’s Group grant of the European Social Funds and the Ministry of Science and Culture of the State of Saxony, Germany, to C.J. and a Young Researcher grant of the Faculty of Medicine of the University of Leipzig to S.R. Competing interests None declared. Ethical approval Obtained from the Ethics Committee at the Faculty of Medicine of the University of Leipzig. Authors’ contributions S.R. designed the study and performed the laboratory experiments together with C.N. M.S. and K.S. provided control data that helped to design the study and to interpret the data. C.C. and I.K. selected the study subjects. C.J. contributed to the study design, data analysis and interpretation, and drafted the manuscript. Acknowledgments We thank R. Schröder for assistance in the recruitment of the subjects and the patients for providing blood specimens. Recombinant HIV gp120 was obtained from GlaxoSmithKline through the National Institute of Biological Standards and Control, programme EVA Centre for AIDS Research. Tetanus toxoid was provided by Behring (now part of Novartis). Hans-Dieter Klenk, Marburg, Germany, provided the influenza virus nucleoprotein gene that was used for the production of recombinant protein. We thank H. Tenckhoff, T. Berg, J. Wiegand and M. Wiese for helpful discussions. References [1] Wahid A, Dubuisson J. Virus-neutralizing antibodies to hepatitis C virus. J Viral Hepat 2013;20(6):369–76.

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