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Human Antibodies 22 (2013) 55–65 DOI 10.3233/HAB-140271 IOS Press

Establishment of human clones producing neutralizing human monoclonal antibodies to the envelope E1/E2 protein of hepatitis C virus by EBV immortalization of immune CD22+ B cells Ashraf Tablla,∗ , Yasmine El Abda , Noha G. Bader El Dina , Reem El Shenawya, Tawfeek H Abdelhafeza, Mostafa El Awadya, Hanan El-Mohamadyb and Sergei Viazovc a

Microbial Biotechnology, National Research Center, Giza, Egypt NAMRU-3, Cairo, Egypt c Institute of virology, Essen, Germany b

Abstract. We aimed to establish Human cell lines producing human monoclonal antibodies to the envelope E1/E2 protein of hepatitis C virus (HCV). Two protocols for EBV immortalization of CD22+ cells separated from HCV positive patients were used; 1) Immortalization with 100% EBV only, 2) immortalization by 30 % EBV and CPG2006. Immortalization was checked microscopically and verified by screening the culture supernatant for antibody production using dot blot and ELISA analysis. ELISA plates were coated by HCV E1/E2 derived from cell lysate transfected by plasmid expressed HCV E1/E2. Also we tested the reactivity of human antibodies based on ELISA plates coating with one linear peptides derived from HCV E1 (a.a 315–319) and two peptides derived from HCV E2 (a.a 412–419) and (a.a 517–530). Neutralization activity was measured using H77C HCV retroviral pseudoparticles (HCVpp). Fifteen clones secreting human immunoglobulin G against HCV E1/E2 protein were isolated. Results of ELISA plates coated with HCV peptides showed that one antibody was binding to E2 peptide (a.a 517–530), and two antibodies binding to HCV E2 peptide (a.a 412–419). The three generated antibodies showed extremely neutralization activity against HCV pp. The three human antibodies were IgG3 and IgG2. These antibodies may be useful for passive immunotherapy of HCV infection Keywords: Human monoclonal antibodies, Hepatitis C virus, EBV, neutralization

1. Introduction Human monoclonal antibodies (hMAb) offer novel ways to probe the B-cell repertoire in health and disease. However, the development of hMAb technology has met with many difficulties due to the instability of the cell lines, the low level of specific antibody se∗ Corresponding author: Ashraf Tabll, Microbial Biotechnology Department, Genetic Engineering and Biotechnology Division, National Research Center, Dokki, 12622, Giza, Egypt. Tel.: +201061216347; Fax: +2023 337 0931; E-mail: ashraftabll@yahoo. com, [email protected].

cretion, and also the poor cloning potency, particularly once using lymphoblastoid cells [1]. However, Martin et al. [2] reported that B lymphocyte immortalization by epstein-barr virus (EBV) is a longtime methodology for antibody production. EBV infects B cells via their CD21 receptor, and afterwards transforms them into frequently dividing, lymphoblastoid cell lines that produce antibodies representing the humoral immune response in vivo. Antibodies are often generated against an infective agent or tumor cells, rendering the resulting antibodies engaging for therapy. The most important advantage of B lymphocyte immortalization, in comparison to different antibody manufacturing tech-

c 2013 – IOS Press and the authors. All rights reserved ISSN 1093-2607/13/$27.50 

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niques [3,4], is that the generation of fully human antibodies that really reflect the specificity and the diversity of the human immune response, generated from the human B lymphocyte repertoire, without the requirement for specific immunization. The first B lymphocyte immortalization technology was performed by culturing B cells within the presence of herpes virus, obtained from the marmoset lymphocyte cell line B958 [5,6]. Numerous changes to the procedure are tried since then, however immortalization and B lymphocyte rate remained inefficient. Recently, Schieffelin et al. [7] reported that human monoclonal antibodies dengue virus envelope (DENV) is with success generated by EBV transformation of B cells from patients at least 2 years after naturally acquired DENV infections. These antibodies show completely different patterns of cross-reactivity, neutralizing, and improvement activity. Schieffelin et al. [7], and others [8, 9] used CpG2006 as stimulators for EBV immortalization. However, antibodies neutralizing SARS corona virus and cytomegalovirus (CMV) were created with success by the introduction of the polyclonal B lymphocyte activator CpG2006 within the B lymphocyte immortalization method or by B lymphocyte activation before EBV infection, respectively [8,9]. Moreover, Fraussen et al. [10] reported that seeding low B lymphocyte numbers per well limits the bias towards the advantageous outgrowth of fast growing immortalized B cells with addition of IL2 and CPG 2006. Nevertheless, Siemoneit et al. [11] used herpes virus immortalization for production of human organism antibodies targeting HCV core macromolecule. Siemoneit et al. [11] represented the institution of 2 vegetative cell lines secreting human monoclonal antibodies to the 22-kD nucleocapsid macromolecule (core, p22) of the viral hepatitis virus (HCV). For this purpose Siemoneit et al., isolated B lymphocytes from associate degree anti-HCV positive donor and infected them with Epstein-Barr (EBV). The B-cell cultures were oligoclonally swollen and 2 of them were coalesced with the (mouse:human) heteromyeloma cell line K6H6/B5. The ensuing stable hybridomas manufacture antibodies of the IgG1/kappa (U1/F10) and also the IgM/kappa (Ul/F11) isotype reacting specifically with the recombinant core macromolecule p22. In the present study, we aimed to the production of Human cell lines manufacturing neutralizing human monoclonal antibodies to the envelope E1/E2 macromolecule of hepatitis C virus (HCV). We used two protocols for EBV immortalization of CD22+ cells separated from HCV positive patients, 1) Immortaliza-

tion with 100% EBV only, 2) immortalization by 30% EBV and CPG2006 with IL2. In this study, 15 human clones producing human antibodies targeting HCV E1/E2 protein were isolated (2 clones from the first protocol and 13 clones from the second protocol). Immortalization was checked microscopically and antibodies production in the culture supernatant was verified using both dot blot and ELISA analysis. Additionally, the targeting epitope for these human antibodies based on ELISA plates coating with one linear peptides derived from HCV E1 (a.a 315–319) and 2 peptides derived from HCV E2 (a.a 412–419) and (a.a 517–530) were tested. Finally, the neutralization activity of the generated human antibodies was measured using HCV pseudo particles (HCV pp).

2. Materials 2.1. Isolation of B-cells (CD22+ ) from PBMCs of HCV positive antibodies patient Informed consent was obtained from HCV positive patient. Then peripheral blood (8–10 ml) was collected from the patient. Peripheral blood mononuclear cells (PBMCs) were purified from heparinized peripheral blood by density gradient centrifugation on Ficoll/Hypaque (Amersham Pharmacia, Milan, Italy). CD22+ lymphocytes were purified by magnetic selection (Miltenyi Biotec, Bologna, Italy) from the PBMCs. Then CD22+ cells were divided into two aliquots that were exposed to immortalization by different EBV-based methods. 2.2. Flow cytometric analysis of CD22+ cells before and after magnetic separation B cells (CD22+ ) were separated by micro-beads (Miltenyi Biotec, Bergisch Gladbach, Germany). To confirm the CD22+ separation we performed immunestaining and flow cytomerty analysis. Direct immunostaining was carried out on ficol separated cells before and after micro beads separation. In brief, 100 µl cell suspension (5000 cells) was incubated with antibodies CD3 FITIC /CD19 PE as double stain (BD Biosciences, San Jose, CA 95131, USA), for 30 min at 8◦ C then washing with PBS/1% FCS two times and finally suspend the pellets in 500 µl PBS until flow cytometric analysis (BD FACSCalibur flow cytometer, San Jose, CA, USA).

A. Tabll et al. / Human clones producing neutralizing human monoclonal antibodies to HCV E1/E2 proteins

2.3. Production of human antibodies 1-Cell immortalization by EBV Propagation of B95.5 (EBV) cell line The marmoset lymphoblastoid cell line B95.8 (kindly provided by Dr. John S Schieffelin, Tulane University School of Medicine, New Orleans, LA, USA) was used as a source of free EBV particles. 2.4. Production of EBV The marmoset lymphoblastoid cell line B95.8, the source of free EBV particles, was grown in DMEM complete medium supplemented with 10% fetal calf serum (FCS) (Gibco, life technologies, NY, USA) and cultured in flasks (Becton Dickinson, NJ, USA) at 37◦ C in a 5% CO2 humidified atmosphere. The cultures were allowed to overgrow by using DMEM with 2% FCS only to encourage virus production. Cultures were harvested and freeze then thawed twice to lyse cells. Cultures were transferred to sterile 50 ml centrifuge tubes and spun at 2000 rpm for 10 min. Supernatants were removed and passed through 0.5 µ m Millipore filters. Filtered supernatant was aliquoted into 1 ml volumes in Nunc cryo tubes and stored at −80◦ C until required. 2.5. Immortalizations of B cells by EBV by two different methods 2.5.1. Immortalizations of B cells by 100% EBV only In the fist protocol, the B cell immortalization was carried out according to Amoli et al. [12]. Briefly, CD22 B cell fractions produced by the Mini-Macs cell separator (MACS) were centrifuged at 2000 rpm for 10 min. Supernatants were carefully aspirated and 2000 µl of EBV particles added to the cell suspension. After careful mixing, 100 µl volumes were transferred into round-bottomed 96 well culture plates (Costar, Corning, NJ, USA). Culture plates were placed in incubator at 37◦ C and 5% CO2 atmosphere for 2 h. A further volume of 100 µl of culture medium (DMEM 20% FCS) was then added to each well and incubated at 37◦ C and 5% CO2 atmosphere. All samples were routinely checked microscopically for cell line transformation. As cells are transformed, they gradually increased in volume then we transferred them to 24 well culture plates (Costar, Corning, NJ, USA) then to Culture flasks (Becton Dickinson, NJ, USA) as reported by Amoli et al. [12]. The cells were routinely checked microscopically every week. Moreover, the cells supernatant were checked after two weeks of EBV transformation using Dot ELISA and ELISA analysis.

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2.5.2. Immortalization of B cells by 30% EBV and CPG2006 with IL2 In the Second protocol, the B cell immortalization was carried according to the method of Fraussen et al. [10]. In brief, CD22 B cells were isolated from PBMC using Mini-Macs cell separator (MACS), then the cells were immortalized by EBV in the presence of B cell stimulates. B cells were cultured for 2 weeks in microtiter plates 96 wells cell culture plates (Costar, Corning, NJ, USA) at 50 cells per well, 1 µg/ml CpG2006 (Invivogen, San Diego Ca, USA) and 30% v/v EBV containing supernatant of the B95-8 cell line. After this immortalization phase, the cells are restimulated during 1 week with 1 µg/ml CpG2006, in combination with 50 U/ml IL-2 (Roch Diganostics, Gmbh, Mannheim, Germany). The culture medium is then replaced and cell cultures are continued without the addition of stimuli for 1 week. Immortalization status is verified after 28 days of culture by performing dot blot analysis for antibody production and by light microscopic examination of cell growth. 2.6. Preparation of cell lysates containing HCV E1/E2 HCV pseudotype particles were used to study the early stages of viral life cycle according to Bartosch et al. [13]; Ashfaq et al. [14]. HCV pp was produced by transfecting three vectors in Human embryo kidney cells (293T). The first vector encodes retroviral Gag and Pol proteins which are responsible for particle budding at the plasma membrane and RNA encapsidation. The second vector encodes a reporter protein (Luciferase). The third vector encodes HCV glycoproteins E1 and E2, which are necessary for viral tropism and fusion of HCV pseudotype particles with target cell membrane. 293T cells secreted virus pseudoparticle with an average 105 particles/ml, which can be used to infect Huh 7 cells and infectivity is evaluated by quantification of the amount of luciferase expressed in Huh-7 cells. Cells were collected after 72 hours from transfection by the Plasmid for HCV E1/E2 and centrifuged at 1200 rpm for 20 min at 8◦ C. However, Dr. Sergie Viazov, one of the co author, and his team work, Essen virology Institute, Germany have used the standard expressing plasmid with the CMV promoter. This plasmed expressed the E1/E2 proteins of the HCV AD78 strain in HEK293 cells and we used the lysate from these cells for ELISA experiments. Then the pellet was incubated with lysing buffer (4 ml tris-HCl, ph.8; 0.8 gm NaCl; 10 ml glycerol; 1 ml triton x-100;

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0.4 ml EDTA and complete to 100 ml with dis.H2 O); then one tablet protease inhibitor (Sigma, USA) was added (one tablet/50 ml lysing buffer). Incubation of cells was carried out on ice for 30 min, then the cells were centrifuged at 1200 rpm for 20 min at 8◦ C. Supernatants were collected and frozen at −80◦ C until use for coating of ELISA plates as reported in Bartosch et al. [13]. 2.7. Dot blot ELISA analysis for human antibody production Culture supernatant (5 µl) of potentially immortalized B cells were spotted on a nitrocellulose blotting membrane Protran BA-85 (VWR International, Chicago, IL, USA). After drying, blots were blocked with 5% (w/v) FCS in PBS (blocking buffer) for 30 min on a shaker. Membranes were then incubated for 1 h with HRP-labelled rabbit anti-human IgG (KPL, Inc, Gaithersburg, Maryland, USA) 1/2500 in blocking buffer and extensively washed in PBS supplemented with 0.05% (v/v) Triton X-100. Antibody production was detected using 3,3 -Diaminobenzidine (DAB) substrate (Sigma Aldrich, St. Louis, MO USA). Human positive HCV sera (in dilutions 1:100; 1:200) were included as positive control. Both culture medium and supernatant from control cells were used as negative control [10]. 2.8. ELISA to test the activity of the generated human antibodies against the HCV E1/E2 transfected cell lysates ELISA plate was coated with lectin in Carbonate buffer and incubated overnight at room Temperature. Then ELISA plate was blocked with blocking buffer and incubated again overnight at 4◦ C. The plate was washed 4 times with PBS T20. Positive HCV E1/E2 cell lysate was added in dilution 1:50 in PBS (100 µl/well) and incubated overnight at 4◦ C. The plate was washed 4 times with PBS then the generated human antibodies supernatant was added 100 µl/ well and culture medium was used as negative control. Also human sera positive for HCV antibodies in dilution 1:100 in dilution buffer were included as positive control and human sera negative for HCV antibodies as another negative control. Then incubation was carried out for 3 hours at 37◦ C. The plate was washed 4 times with PBS T20 and Conjugate antihuman peroxidase (KPL, Inc, Gaithersburg, Maryland, USA) (1:5000) in dilution buffer was added (100 µl/

well) and incubated for 1 hour at 37◦ C. The plate was washed 4 times with PBS then the substrate; one tablet OPD/10 ml PBS with 10 µl H2 O2 was added (100 µl/ well) and incubated at RT for 5–15 minutes. Color was read by ELISA reader at 450 nm (Tecan; Sunrise, Austria, GmbH). However, this assay were used to test sera samples positive for HCV antibodies with genotype 4a, the predominate type in Egypt, and compare it with sera samples from Germany with genotype 2a. All sera samples positive for HCV antibodies/HCV RNA with either genotype 4a or genotype 2a showed reactivity in this assay 2.9. ELISA to test the activity of the generated human antibodies against linear synthetic HCV E1 and E2 peptides Enzyme linked immunosorbent assay (ELISA) was established in house. Briefly, polystyrene micro titer ELISA plates were coated with 50 µl/well of (5 ng/ml) of synthetic peptides derived from HCV E1 P315 (a.a. 315–323) and two peptides from E2 region designated p517 (a.a. 517–530) and p412 (a.a. 412–419) diluted in carbonate/ bicarbonate buffer (pH 9.6). These peptides were synthesized commercially by ANASPEC, Inc, (San Jose CA, USA). The plates were incubated overnight at room temperature and washed three times using 0.05% (v/v) PBS-T20 (pH 7.2). Free active sites were blocked using 0.2% (w/v) nonfat milk in carbonate/bicarbonate buffer. After washing, 50 µl/well from each culture supernatant containing human antibodies were added, and incubated at 37◦ C for 2 h. Media were used as negative control. Also human sera positive for HCV antibodies were used as positive control and human sera negative for HCV antibodies as another negative control. After washing, 50 µl/well of antihuman IgG peroxidase conjugate (KPL, Inc, Gaithersburg, Maryland, USA), diluted in 0.2% (w/v) nonfat milk in PBS-T2 were added and the mixture was incubated at 37◦ C for 1 h. The amount of coupled conjugate was determined by incubation with 50 µl/well O-Phenylene Diamine (OPD, 0.01%) substrate (Sigma Aldrich, St. Louis, MO USA) for 30 min at 37◦ C. Finally, the reaction was stopped using 3 M HCl and the absorbance was read at 450 nm (Tecan; Sunrise, Austria, GmbH). 2.10. Detection of IgG subclass of the generated human antibodies IgG subclass of the generated human antibodies was determined by commercial kit according to the in-

A. Tabll et al. / Human clones producing neutralizing human monoclonal antibodies to HCV E1/E2 proteins

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Fig. 1. Flow cytometric analysis of cells before and after magnetic Separation. Results of flow cytometric analysis showed increase and concentration of B cells after magnetic separation than before magnetic separation. (Colours are visible in the online version of the article; http://dx.doi. org/10.3233/HAB-140271)

struction of manufacture (Invitrogen Corporation, CA, USA). The Human IgG subclass profile ELISA Kit contains components required to construct an enzymelinked immunoassay for the specific and quantitative measurement of human IgG1, IgG2, IgG3, and IgG4 subclasses. 2.11. Neutralization assay using HCV pp system HCV pseudotype particles were used to study the early stages of viral life cycle. This virus like particles was used for testing the neutralizing activity of the generated human antibodies. In brief, HCVpp infect Huh 7 cells and infectivity is evaluated by quantification of the amount of luciferase expressed in Huh-7 cells. To measure luciferase activity, Huh 7 cells were washed twice with PBS and lysed with 100 µl cell culture lysis reagent (Promega, Madison, WI, USA) according to the manufacturer’s recommendations. Luciferase activity was measured with the luciferase assay system (Promega, Madison, WI, USA) using a Lumat LB9507 luminometer (EG & G Berthold, Bad Wildbad, Germany).

3. Results 3.1. Separation of B cells (CD22+ ) The separated B-cells (responsible for production of human antibodies) were tested by immune-staining

and flow cytometry analysis. CD22+ cells were isolated from human PBMCs using CD22+ micro beads, MS column and min MACs separator. The separated cells were flourescently stained with CD19PE/CD3 FITC. As shown in Fig. 1(B) cells (CD22+ ) recorded higher percentage after micro beads separation than before separation. 3.1.1. Immortalization and propagation of CD22+ cells by 100% EBV only The immortalized cells by 100% EBV were followed up and checked microscopically using inverted microscope and the growth of cell clones started to appear after 3 days. No growth of cells (CD22+ cells) without EBV. Figure 2(a) showed the presence of growing clones in the wells containing cells with EBV while no growth was observed in control cells without EBV after one week of EBV immortalization. 3.1.2. Immortalization and propagation of CD22+ cells by EBV (30%), CPG2006 and IL2 The immortalized cells by EBV (30%), CPG2006 and IL2 were followed up and checked microscopically using inverted microscope and the growth of cell clones started to appear after 3 days. Figure 2(b) showed different immortalized and growth of cell clones in wells B, C, D while no growth in control cells without EBV after one and two weeks of started culture. Moreover, higher growth rate of immortalized cells were observed after two week compared to one week after cell immortalization.

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Fig. 2c. Comparison between the two protocols of cell immortalization larger cell clones were noticed in cell immortalization with 100% EBV more than cell immortalization with 30% EBV with CPG2006/IL2.

Fig. 2a. Cell immortalization with EBV after one week and after two weeks (following up the same wells after one week and after two weeks) A: control wells. Separated CD22+ cells propagated in media only without EBV. B, C, D different wells containing immortalized cell clones after one and two weeks.

duced by 30% EBV with CPG2006/IL2. Figure 3 showed the growth of human clones with different magnification showed the cluster (clumps shapes) of human monoclonal cells after seeding it in 12 wells cell culture plates. 3.2. Dot blot analysis for human antibody production To confirm the production of antibody inside the immortalized cells dot blot analysis was performed. Figure 4 showed dark brown spot in positive HCV human sera while no spot was observed in culture medium or media supernatant of negative control cells. Supernatant from different wells of immortalized cells showed dark brown spot, which indicated that immortalized cells secreted human antibodies. 3.3. Screening of human antibodies against HCV E1/E2 cell lysate by ELISA

Fig. 2b. Cell immortalization with EBV 30% with CPG2006 and IL2 after one week and two weeks. The wells A, B, C and D were followed after one week and two weeks).

3.1.3. Comparison between the two protocols of cell immortalization Immortalization was checked microscopically. We noticed large size of cell clones produced by immortalization with 100% EBV only compared to that pro-

In the immortalization of separated CD22+ by EBV (30%) with CPG2006 and IL2 protocol; 13 clones were isolated and showed high reactivity with positive HCV E1/E2 cell lysate using ELISA. We focused only on the highly optical density clones. Negative control of the experiment showed no reactivity, while positive control showed high optical density as shown in the Fig. 5(a). While, in the immortalization of separated CD22+ by EBV(100%) protocol; 2 clones only were isolated and showed high reactivity with positive HCV E1/E2 cell lysate using ELISA as shown in Fig. 5(b). Also we noticed that the cell immortalized with EBV (30%) with CPG2006 and IL2 showed very high optical den-

A. Tabll et al. / Human clones producing neutralizing human monoclonal antibodies to HCV E1/E2 proteins

Fig. 3. Different view of human monoclonal cells on inverted light microscope with different magnification showed the cluster (clumps shapes) of human monoclonal cells after seeding it in 12 wells cell culture plates.

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Fig. 5. (a) Detection of human antibodies in the cell supernatant secreted by growth clones and measured by ELISA after two weeks of cell. Immortalization by EBV 30% with CPG2006 and IL2; (b) Detection of human antibodies in the cell supernatant secreted by growth clones and measured by ELISA after two weeks of cell immortalization by EBV only.

IgG3 while one antibody was IgG1. Other antibodies were mixed isotypes particularly isotypes 3 and 2. 3.5. Screening of human antibodies against HCV E1/E2 synthetic peptides by ELISA

Fig. 4. Dot blot analysis for antibody production after two weeks of B cell immortalization by both EBV protocols. (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/ HAB-140271)

sity than the immortalization by EBV only this means that addition of cell stimulator such as CPG2006/IL2 is very important for stimulating B cells to produce antibodies.

Reactivity of the generated human antibodies against peptides derived from HCV E1 (a.a 315–323), HCV E2 (a.a 412–419) and HCV E2 (a.a 517–531) was measured using ELISA. As shown in Fig. 6(a,b), arrows indicated the reactivity of antibodies # 11, 12 against peptide E2 (a.a 412–419). Arrow indicates to reactivity of human monoclonal antibodies Ab # 9 with synthetic peptides derived from HCV E2 (a.a 517–531). While human antibodies # 4, 5, 6, 7, and 8 (star) show reactivity against all tested peptides from HCV E1 and HCV E2.

3.4. IgG isotyping of the generated human antibodies

3.6. Neutralizing activities of the generated human antibodies using HCV pp system

Human IgG isotyping was measured by commercial ELISA kit. We found two antibodies were isotype

Effect on virus entry was measured using H77C HCV retroviral pseudoparticles, HCVpp. As shown in

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(a)

Fig. 7. Neutralizing activities of the generated human antibodies based on HCV pp system.

(b)

Fig. 6. (a) Reactivity of the generated human antibodies against peptides derived from HCV E1 (a.a 315–323), HCV E2 (a.a 412–419) and HCV E2 (a.a 517–531) as measured my ELISA. Arrow indicated that the antibody binds to one peptide only Star indicated antibody binds to two or three linear peptides; (b) Continuous and discontinuous epitopes. Abs # 9, 11, 12 binds only with continuous epitope while Abs # 4, 5, 6, 7, 8 binds with discontinuous epitopes. (Colours are visible in the online version of the article; http:// dx.doi.org/10.3233/HAB-140271)

Fig. 7, we found three human antibodies # 1, 8, 9 were neutralizing antibodies using HCV pp system where the other antibodies were failed to neutralize HCV pseudo particles. However, antibodies # 14, 15 generated by 100% EBV failed to neutralize HCV pp while one Ab # 15 neutralized HCV cc (data not shown).

4. Discussion Production of human monoclonal antibodies can be carried out by different methods such as phage display, humanization of mouse monoclonal antibodies, by transgenic mice or recently by immortalization of B-cells. Recently, several authors reported several studies on the immortalization of B cells [10,12,15, 16]. In the present study, we established cell lines producing human monoclonal antibodies targeting HCV E1/E2 epitopes. Firstly, B cells were isolated from peripheral blood mononuclear cells by micro beads conjugated with monoclonal antibodies targeting hu-

man CD22+ . Then, the separation of CD22+ by micro beads was confirmed by immune-staining of B cells and analysis by flow cytometric Fig. 1. Secondly, the B cells (CD22+ ) were immortalized using two protocols. In the first protocol the B cells were immortalized with 100% EBV as reported by Amoli et al. [12] while in the second protocol the B cells immortalization by 30% EBV and CPG2006 with IL2 reported recently by Fraussen et al. [10]. However in the study of Amoli et al. [12], they reported that EBV immortalized B lymphocyte cell lines have been extensively used as a source of biological material for functional and molecular studies and represent a potentially limitless source of genomic DNA but in the present study we try to use the same protocol for study of production of human monoclonal antibodies. Both Immortalization protocol produced human antibodies as verified by Dot ELISA. The results of ELISA coated by cell lysate positive for HCV E1/E2 showed reactivity for two clones only from 100% EBV immortalization protocol versus 13 clones from the 30% EBV, CpG2006 and IL-2 immortalization protocol. Moreover, the results showed higher optical density of the 13 clones secreted against positive HCV E1/E2 cell lysate which indicate the production of highly reactive antibodies. The results showed that immortalization by EBV 100% only produces large clones Fig. 2 compared to immortalization by 30% EBV and CPG2006 with IL2 but the antibodies level, as measured by ELISA, showed high optical density in cells immortalized with 30% EBV and stimulant CPG2006 and IL2 and this indicated the role of cell stimulators for production of antibodies. Also, the results indicate that immortalization of low B cell number by 30% EBV, CPG2006 and IL2 was introduced with high efficiency and reproducibility for low B cell numbers that makes immediate generation

A. Tabll et al. / Human clones producing neutralizing human monoclonal antibodies to HCV E1/E2 proteins

of single clones producing monoclonal antibodies. Our results confirmed the results of Fraussen et al. [12] in which low number of B cells with CPG 2006 and IL2 stimulators produced monoclonal antibodies. Neutralizing antibodies have vital role in control of HCV infection [17]. Lavillette et al. [18] and Pestka et al. [19] have shown that neutralizing antibodies are induced within the early phase of infection by patients who after infection or resolve infection. The E1/E2 envelope surface glycoprotein of HCV (HCVE1/E2) is that the main target of the host humoral immune response and for this reason one of the main variable viral proteins. Broadly cross-neutralizing monoclonal antibodies directed against HCVE1 /E2 represent a promising tool for the study of virus-host interaction and for the development of effective prophylactic and therapeutic approaches. In the last few years several anti-HCV/E2mAbs are evaluated in preclinical and clinical trials as potential candidate antiviral therapy for administration in pre- and post-transplant settings [17]. In the present study, we used linear peptides derived from HCV E1 (a.a 315–323), and 2 peptides from HCV E2 (a.a 412–419) and HCV E2 (a.a 517– 531) in house optimized based ELISA assay to check the targeting epitope of the generated antibodies and these regions were studied by several investigators [17, 20–22]. The results of in house based ELISA using the synthetic peptides indicated the reactivity of two antibodies (Ab # 11, 12) against peptide E2 (a.a 412–419) and one antibody (Ab # 9) against of HCV E2 (a.a 517– 531). While five human antibodies (Ab # 4, 5, 6, 7 and 8) showed reactivity against the three tested HCV E1 and HCV E2 peptides. So in our opinion we think that human antibodies # 4, 5, 6, 7 and 8 may be binding with a discontinuous epitope based on ELISA results and this point requires further studies. However, discontinuous epitope consists of a group of residues that are not contiguous within the sequence, however are brought together by the folding of the polypeptide sequences, or by the juxtaposition of two separate peptide chains. Also, recloning by serial dilution methodology and testing by ELISA once more ought to be applied to get to make sure that these clones are monoclonal. i.e. are not still multiple human clones. Recently Giang et al. [23] isolated seventy three human mAbs recognizing 5 distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV immune phage-display antibody library by using an exhaustive panning strategy. Several of those mAbs were broadly neutralizing. Additionally Giang et al. [23] reported that human antibody AR4A, rec-

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ognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has broadly neutralizing activity toward various HCV genotypes. Moreover, the results of the neutralization assay using the HCV pp should that three antibodies have neutralizing activity. Neutralizing antibodies are vital for inhibition of viral entry and post binding entry of the virus to the target cells [24]. Zhang et al. [25,26] represented an extra escape whereby the presence of non neutralizing antibodies interferes with the performance of neutralizing antibodies, leading to the reduction or blockage of their effect. Two epitopes of HCV E2 were mapped epitope I (a.a 412–419) has been recognized as a very important neutralization site, while epitope II (a.a 434– 446) showed interference with antibody to epitope I inhibiting neutralization of the virus [25]. Epitope Iand epitope II- specific antibodies were detected in plasma from chronically HCV-infected patients. Moreover, several authors described several monoclonal antibodies targeting aa 412–423 (epitope I) and it is function as CD81 binding [24,27,28]. This region was established to play an important role in forming complexes of E2 with CD81 [29] and have a direct role in entry by mediating CD81 binding [30]. Furthermore, murine mAbs (AP33 and 3/11) that recognized a peptide defined as amino acids 412–423 (known as epitope I) of the HCV polyprotein were found to efficiently neutralize HCV viral entry [25–27]. However, two of our human monoclonal antibodies Abs # 11, 12 bind with linear peptides of HCV E2 at a.a 412–419. We think this study needs further more investigation on these antibodies such as crystallography x ray analysis of the antibodies and its epitope. Also another neutralization assays are required to confirm the activity of these antibodies. In conclusion, the produced human antibodies can be useful for the development of effective prophylactic and therapeutic approaches against HCV infection.

Acknowledgements The marmoset lymphoblastoid cell line B95.8 (kindly provided by Dr. John S Schieffelin, Tulane University School of Medicine, New Orleans, LA, USA) was used as a source of free EBV particles. Authors thank Sina Luppus for here assistant. This work was funded through the Science and Technology Development Fund (STDF), Egypt-grant no. 894” to Dr. Ashraf Tabll.

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