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ABSTRACT: A challenge in hepatitis C virus (HCV) vaccine development is defining conserved protective epitopes. A cluster of these epitopes comprises an immunodominant domain on the E2 glycoprotein, designated domain B. CBH-2 is a neutralizing human monoclonal antibody to a domain B epitope that is highly conserved. Alanine scanning demonstrated that the epitope involves residues G523, G530, and D535 that are also contact residues for E2 binding to CD81, a coreceptor required for virus entry into cells. However, another residue, located at position 431 and thus at a considerable distance in the linear sequence of E2, also contributes to the CBH-2 epitope. A single amino acid substitution at this residue results in escape from CBH-2-mediated neutralization in a genotype 1a virus. These results highlight the challenges inherent in developing HCV vaccines and show that an effective vaccine must induce antibodies to both conserved and more invariant epitopes to minimize virus escape.
Journal of Virology 07/2008; 82(12):6067-72. · 5.40 Impact Factor
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ABSTRACT: The humoral response to hepatitis C virus (HCV) may contribute to controlling infection. We previously isolated human monoclonal antibodies to conformational epitopes on the HCV E2 glycoprotein. Here, we report on their ability to inhibit infection by retroviral pseudoparticles incorporating a panel of full-length E1E2 clones representing the full spectrum of genotypes 1-6. We identified one antibody, CBH-5, that was capable of neutralizing every genotype tested. It also potently inhibited chimeric cell culture-infectious HCV, which had genotype 2b envelope proteins in a genotype 2a (JFH-1) background. Analysis using a panel of alanine-substitution mutants of HCV E2 revealed that the epitope of CBH-5 includes amino acid residues that are required for binding of E2 to CD81, a cellular receptor essential for virus entry. This suggests that CBH-5 inhibits HCV infection by competing directly with CD81 for a binding site on E2.
Journal of General Virology 04/2008; 89(Pt 3):653-9. · 3.36 Impact Factor
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ABSTRACT: Human monoclonal antibodies derived from B cells of HCV-infected individuals provide information on the immune response to native HCV envelope proteins as they are recognized during infection. Monoclonal antibodies have been useful in the determination of the function and structure of specific immunogenic domains of proteins and should also be useful for the structure/function characterization of HCV E1 and E2 envelope glycoproteins. The HCV E2 envelope glycoprotein has at least three immunodistinctive conformation domains, designated A, B, and C. Conformational epitopes within domain B and C are neutralizing antibody targets on HCV pseudoparticles as well as from infectious cell culture virus. In this study, a combination of differential surface modification and mass spectrometric limited proteolysis followed by alanine mutagenesis was used to provide insight into potential conformational changes within the E2 protein upon antibody binding. The arginine guanidine groups in the E2 protein were modified with CHD in both the affinity bound and free states followed by mass spectrometric analysis, and the regions showing protection upon antibody binding were identified. This protection can arise by direct contact between the residues and the monoclonal antibody, or by antibody-induced conformational changes. Based on the mass spectrometric data, site-directed mutagenesis experiments were performed which clearly identified additional amino acid residues on E2 distant from the site of antibody interaction, whose change to alanine inhibited antibody recognition by inducing conformational changes within the E2 protein.
Biochimica et Biophysica Acta 04/2008; 1784(3):530-42. · 4.66 Impact Factor
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ABSTRACT: Development of full-length hepatitis C virus (HCV) RNAs replicating efficiently and producing infectious cell-cultured virions, HCVcc, in hepatoma cells provides an opportunity to characterize immunogenic domains on viral envelope proteins involved in entry into target cells. A panel of immunoglobulin G1 human monoclonal antibodies (HMAbs) to three immunogenic conformational domains (designated A, B, and C) on HCV E2 glycoprotein showed that epitopes within two domains, B and C, mediated HCVcc neutralization, whereas HMAbs to domain A were all nonneutralizing. For the neutralizing antibodies to domain B (with some to conserved epitopes among different HCV genotypes), the inhibitory antibody concentration reducing HCVcc infection by 90%, IC90, ranged from 0.1 to 4 microg/ml. For some neutralizing HMAbs, HCVcc neutralization displayed a linear correlation with an antibody concentration between the IC50 and the IC90 while others showed a nonlinear correlation. The differences between IC50/IC90 ratios and earlier findings that neutralizing HMAbs block E2 interaction with CD81 suggest that these antibodies block different facets of virus-receptor interaction. Collectively, these findings support an immunogenic model of HCV E2 having three immunogenic domains with distinct structures and functions and provide added support for the idea that CD81 is required for virus entry.
Journal of Virology 02/2007; 81(2):1043-7. · 5.40 Impact Factor
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Rachel Eren,
Dorit Landstein,
Dov Terkieltaub,
Ofer Nussbaum,
Arie Zauberman,
Judith Ben-Porath,
Judith Gopher,
Rachel Buchnick,
Riva Kovjazin,
Ziva Rosenthal-Galili, [......],
Ofer Ben-Moshe,
Alberto Kischitsky, Steven K H Foung,
Zhen-Yong Keck,
Orit Pappo,
Ahmed Eid,
Oded Jurim,
Gidi Zamir,
Eithan Galun,
Shlomo Dagan
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ABSTRACT: Passive immunotherapy is potentially effective in preventing reinfection of liver grafts in hepatitis C virus (HCV)-associated liver transplant patients. A combination of monoclonal antibodies directed against different epitopes may be advantageous against a highly mutating virus such as HCV. Two human monoclonal antibodies (HumAbs) against the E2 envelope protein of HCV were developed and tested for the ability to neutralize the virus and prevent human liver infection. These antibodies, designated HCV-AB 68 and HCV-AB 65, recognize different conformational epitopes on E2. They were characterized in vitro biochemically and functionally. Both HumAbs are immunoglobulin G1 and have affinity constants to recombinant E2 constructs in the range of 10(-10) M. They are able to immunoprecipitate HCV particles from infected patients' sera from diverse genotypes and to stain HCV-infected human liver tissue. Both antibodies can fix complement and form immune complexes, but they do not activate complement-dependent or antibody-dependent cytotoxicity. Upon complement fixation, the monoclonal antibodies induce phagocytosis of the immune complexes by neutrophils, suggesting that the mechanism of viral clearance includes endocytosis. In vivo, in the HCV-Trimera model, both HumAbs were capable of inhibiting HCV infection of human liver fragments and of reducing the mean viral load in HCV-positive animals. The demonstrated neutralizing activities of HCV-AB 68 and HCV-AB 65 suggest that they have the potential to prevent reinfection in liver transplant patients and to serve as prophylactic treatment in postexposure events.
Journal of Virology 04/2006; 80(6):2654-64. · 5.40 Impact Factor
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ABSTRACT: Hepatitis C (HCV) E2 glycoprotein is involved in virus attachment and entry, and its structural organization is largely unknown. Characterization of a panel of human monoclonal antibodies (HMAbs) to HCV by competition studies has led to an immunogenic organization model of E2 with three domains designated A, B, and C and epitopes in each domain having similar structural and functional properties. Domain A contains nonneutralizing epitopes, and domains B and C contain neutralizing epitopes. The isolation and characterization of three new HMAbs within domain A for a total of six provide support for this model. All six domain A HMAbs do not neutralize HCV retroviral pseudotype particle (HCVpp) infection on Huh-7 cells, and all six HMAbs have similar binding affinity and maximum binding, B(max), a relative indicator of epitope density, as other neutralizing HMAbs, suggesting that neutralization is epitope specific and not by binding to any surface epitope. The dose-dependent neutralizing activity of CBH-7, an HMAb to a domain C epitope in spatial proximity to domain A, and of CBH-5, a domain B HMAb to a more distant epitope, were tested in the presence and absence of each domain A HMAb. No enhancement or reduction in CBH-7 or CBH-5 neutralizing activity was observed, indicating that the potential induction of nonneutralizing antibodies should not be a central issue for HCV vaccine design. To assess whether domain A is involved in the structural changes as part of a pH-dependent virus envelope fusion process, changes in antibody binding patterns to normal pH and acid pH-treated HCVpp were measured. Antibody binding affinity of HMAbs to HCVpp was not affected by low pH. However, the B(max) values for low-pH-treated HCVpp with antibodies to domain A increased 46%, for domain C (CBH-7) they increased 23%, and for domain B (CBH-5) there was a decrease of 12%. Collectively, the organization and function of HCV E2 antigenic domains are roughly analogous to the large envelope glycoprotein E organizational structure for other flaviviruses with three distinct structural and functional domains.
Journal of Virology 12/2005; 79(21):13199-208. · 5.40 Impact Factor
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ABSTRACT: The recombinant E1E2 heterodimer of the hepatitis C virus is a candidate for a subunit vaccine. Folding analysis of E1 and E2 glycoproteins, stably expressed in CHO cells, showed that E1 folding was faster and more efficient than E2. The oxidized DTT-resistant conformation of E1 was completed within 2 h post-synthesis, while E2 not only required up to 6 h but also generated non-native species. Calnexin was found to assist E1 folding, whereas no chaperone association was found with E2. The assembly of E1 and E2 was assessed by co-immunoprecipitation and sedimentation velocity analysis. We found that the formation of native E1E2 heterodimers paralleled E2 oxidation kinetics, suggesting that E2 completed its folding process after association with E1. Once formed, sedimentation of the native E1E2 heterodimers was consistent with the absence of additional associated factors. Taken together, our data strongly suggest that productive folding of the major HCV spike protein E2 is assisted by E1.
Virology 03/2005; 332(1):438-53. · 3.35 Impact Factor
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ABSTRACT: A panel of human monoclonal and recombinant antibody light chains was screened for cleavage of the synthetic peptide corresponding to a neutralizing epitope of hepatitis C virus (residues 192-205 of envelope glycoprotein E1). One of the 39 light chains studied hydrolyzed the Val197-Ser198 bond of the peptide with Km and kcat values of 223 +/- 7 microM and 0.087 +/- 0.001 min(-1).
Bioorganic & Medicinal Chemistry Letters 10/2004; 14(17):4529-32. · 2.55 Impact Factor
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ABSTRACT: Mechanisms of virion attachment, interaction with its receptor, and cell entry are poorly understood for hepatitis C virus (HCV) because of a lack of an efficient and reliable in vitro system for virus propagation. Infectious HCV retroviral pseudotype particles (HCVpp) were recently shown to express native E1E2 glycoproteins, as defined in part by HCV human monoclonal antibodies (HMAbs) to conformational epitopes on E2, and some of these antibodies block HCVpp infection (A. Op De Beeck, C. Voisset, B. Bartosch, Y. Ciczora, L. Cocquerel, Z. Y. Keck, S. Foung, F. L. Cosset, and J. Dubuisson, J. Virol. 78:2994-3002, 2004). Why some HMAbs are neutralizing and others are nonneutralizing is looked at in this report by a series of studies to determine the expression of their epitopes on E2 associated with HCVpp and the role of antibody binding affinity. Antibody cross-competition defined three E2 immunogenic domains with neutralizing HMAbs restricted to two domains that were also able to block E2 interaction with CD81, a putative receptor for HCV. HCVpp immunoprecipitation showed that neutralizing and nonneutralizing domains are expressed on E2 associated with HCVpp, and affinity studies found moderate-to-high-affinity antibodies in all domains. These findings support the perspective that HCV-specific epitopes are responsible for functional steps in virus infection, with specific antibodies blocking distinct steps of virus attachment and entry, rather than the perspective that virus neutralization correlates with increased antibody binding to any virion surface site, independent of the epitope recognized by the antibody. Segregation of virus neutralization and sensitivity to low pH to specific regions supports a model of HCV E2 immunogenic domains similar to the antigenic structural and functional domains of other flavivirus envelope E glycoproteins.
Journal of Virology 10/2004; 78(17):9224-32. · 5.40 Impact Factor
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ABSTRACT: Human antibodies elicited in response to hepatitis C virus (HCV) infection are anticipated to react with the native conformation of the viral envelope structure. Isolation of these antibodies as human monoclonal antibodies that block virus binding and entry will be useful in providing potential therapeutic reagents and for vaccine development. H-111, an antibody to HCV envelope 1 protein (E1) that maps to the YEVRNVSGVYH sequence and is located near the N terminus of E1 and is able to immunoprecipitate E1E2 heterodimers, is described. Binding of H-111 to HCV E1 genotypes 1a, 1b, 2b, and 3a indicates that the H-111 epitope is highly conserved. Sequence analysis of antibody V regions showed evidence of somatic and affinity maturation of H-111. Finally, H-111 blocks HCV-like particle binding to and HCV virion infection of target cells, suggesting the involvement of this epitope in virus binding and entry.
Journal of Virology 08/2004; 78(13):7257-63. · 5.40 Impact Factor
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ABSTRACT: Little is known about the role of the humoral immune response to hepatitis C virus (HCV). This study provides molecular evidence for the mechanism by which neutralizing Abs from the sera of chronic HCV patients have lower inhibitory activities against the binding of HCV E2 envelope protein to human hepatoma cell lines than to a lymphoma cell line. E2 binds to several putative receptors, specifically human CD81; human scavenger receptor, class B, type 1; and heparan sulfate. We have shown that E2 binds to target cells via these receptors in a noncompetitive manner. Thus, incomplete inhibition of one of the receptors leads to only a partial E2 blockade and, possibly, evasion of the host immune response. We demonstrated that the difference in and reduction of inhibition was closely related to impaired blockade of E2 binding to scavenger receptor, class B, type 1, and heparan sulfate. We have also shown that soluble E2 protein binds to multiple soluble receptors via separate binding domains on E2, providing further evidence for the distinct recognition of multiple cellular receptors by E2. This report suggests a novel finding that biased humoral immune responses to HCV E2 might provide an alternative mechanism for viral escape without the involvement of mutation. Additionally, our data give crucial consideration to the development of HCV vaccines that stimulate protective humoral immune responses.
The Journal of Immunology 08/2004; 173(1):446-55. · 5.79 Impact Factor
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ABSTRACT: The majority of hepatitis C virus (HCV)-infected individuals progress from acute to chronic disease, despite the presence of a strong humoral immune response to the envelope glycoproteins E1 and E2. When expressed in mammalian cells, E1 and E2 form both noncovalently linked E1E2 heterodimers, believed to be properly folded, and disulfide-linked, high-molecular-weight aggregates that are misfolded. Previously, we identified 10 human monoclonal antibodies (HMAbs) that bind E2 glycoproteins from different genotypes. Here we demonstrate that one of these HMAbs, CBH-2, is unique in its ability to distinguish between properly folded and misfolded envelope proteins. This HMAb recognizes HCV-E2 only when complexed with E1. The E1E2 complexes recognized by CBH-2 are noncovalently linked heterodimers and not misfolded disulfide-linked, high-molecular-weight aggregates. The E1E2 heterodimers seen by CBH-2 no longer associate with the endoplasmic reticulum chaperone calnexin and are likely to represent the prebudding form of the HCV virion.
Journal of Virology 02/2003; 77(2):1604-9. · 5.40 Impact Factor
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ABSTRACT: The envelope glycoprotein E2 of hepatitis C virus (HCV) is a major component of the viral envelope. Knowledge of its topologic features and antigenic determinants in virions is crucial in understanding the viral binding sites to cellular receptor(s) and the induction of neutralizing antibodies. The lack of a robust cell culture system for virus propagation has hampered the characterization of E2 presented on the virion. Here we report the structural features of hepatitis C virus-like particles (HCV-LPs) of the 1a and 1b genotypes as determined by various mouse and human monoclonal anti-envelope antibodies. Our results show that the E2 protein of HCV-LPs reacts with human monoclonal antibodies recognizing conformational determinants. Monoclonal antibodies (mAbs) specific for the hypervariable region 1 (HVR-1) sequence reacted strongly with HCV-LPs, suggesting that the HVR-1 is exposed on the viral surface. Several mAbs recognized both HCV-LPs with equally high affinity, indicating that the corresponding epitopes [amino acids (aa) 192-217 of E1 and aa 412-423, aa 522-531, and aa 640-653 of E2] are conserved in both genotypes and exposed on the surface of the HCV-LP. The E2 and E1/E2 dimers of 1a bound strongly to the recombinant large extracellular loop (LEL) of CD81 (CD81-LEL) of human and African green monkey, while the HCV-LP of 1a bound weakly to human CD81-LEL. E1/E2 dimers and the HCV-LPs of 1b did not bind CD81-LEL, consistent with the notion that CD81 recognition by E2 is strain-specific and does not correlate with permissiveness of infection. A model of the topology and exposed antigenic determinants of the envelope proteins of HCV is proposed.
Virology 07/2002; 298(1):124-32. · 3.35 Impact Factor
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ABSTRACT: The majority of the antibody response to HTLV-1 surface glycoprotein, gp46, is directed at conformational epitopes. However, the regions of HTLV-1 gp46 that contain conformational epitopes are poorly defined. We previously reported on human monoclonal antibodies (hMAbs) to conformational epitopes within the HTLV-1 surface glycoprotein (gp46) that inhibit HTLV-1-mediated syncytium formation (Hadlock KG, Rowe J, Perkins S, et al.: J Virol 1997;71:5828-5840). To localize the conformational epitopes recognized by these antibodies, chimeric envelope proteins were constructed in which selected regions of the HTLV-1 envelope were replaced with the corresponding sequences from other members of the HTLV family of retroviruses. The chimeras were tested for reactivity with three hMAbs to conformational epitopes in HTLV-1 gp46, PRH-7A, PRH-3, and PRH-4, and one hMAb to a linear epitope, 0.5alpha. hMAb PRH-3 was specifically nonreactive with a chimera that replaced amino acids 32-36 of HTLV-1 gp46 and exhibited sharply reduced reactivity with a chimera that replaced amino acids 224-251 of HTLV-1 with the corresponding HTLV-2 sequence. hMAb PRH-4 was specifically nonreactive with a construct replacing amino acids 1-162 of HTLV-1 gp46 with the corresponding HTLV-2 sequence. Thus, HTLV-1 gp46 contains multiple conformational epitopes located in the amino-terminal portion of the protein.
AIDS Research and Human Retroviruses 02/2002; 18(1):57-70. · 2.25 Impact Factor
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ABSTRACT: The genotype of hepatitis C virus (HCV) can profoundly affect the success of antiviral therapy for HCV infection. A possible contributing factor is a varied immune response elicited by infection with different HCV genotypes. In this study, full-length E2 proteins of HCV genotypes 1a, 1b, 2a, and 2b were used to determine the fraction of the humoral immune response to HCV E2 that is genotype specific. Greater than 90% of all infected individuals had serum antibodies to the four E2 proteins. Overall, individuals infected with genotype 1a or 1b were characterized by variable immune responses to HCV E2 with relatively high amounts of cross-reactivity with other E2 proteins. Individuals infected with genotype 2a or 2b exhibited a strong preferential reactivity to genotype 2a and 2b E2 proteins. Individuals with elevated titers to HCV E2 were more likely to be infected with genotype 2a and had a significantly lower median viral load. These findings indicate that the antibody response to HCV E2 is affected by the genotype of the virus and that induction of a strong humoral immune response to HCV E2 may contribute to a decreased viral load. J. Med. Virol. 65:23–29, 2001. © 2001 Wiley-Liss, Inc.
Journal of Medical Virology 07/2001; 65(1):23 - 29. · 2.82 Impact Factor
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ABSTRACT: The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.
Journal of Virology 12/2000; · 5.40 Impact Factor
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ABSTRACT: Epstein Barr virus (EBV) can be used to transform human B-lymphocytes to derive populations of cells secreting specific antibodies
of interest. Isolating monoclonal or stable populations of these cells, however, has proven very difficult (1). In our laboratory, we have developed methods to immortalize specific antibody-producing cells by fusing secreting EBV-activated
lymphocytes to mouse-human heteromyeloma cell lines with electrofusion, followed by cloning (2). This methodology has allowed us to produce human hybridomas secreting 1–200 µg/mL of IgG specific for HCMV (3), HTLV-I (4), and HCV (unpublished) using several different mouse-human heteromyeloma fusion partners. Because as few as 5 × 104–106 EBV-activated B-cells can be successfully fused with a high degree of efficiency and consistency (up to one hybrid for each
100–1000 input EBV-activated cells), they can be fused as soon as antibody can be detected in a microtiter well, before the
cells lose secretion or are overgrown by nonsecreters (5). High efficiency is achieved by varying the electrical parameters depending on the specific cells, the cell number, and
the medium in which the cells are fused (see
Table 1).
Table 1Examples of Fusion Voltage Used with Different Fusion Partners in Different Fusion Media to Immortalize Antigen-Specific Antibody
Secreted by EBV-Activated Lymphocytes from Peripheral Blood
Hybrid name
Antibody secreted to
IgG, µg/mL
# EBV fused
Heteromyeloma fusion partner
Fusion medium
dc Fusion voltage
Range in hybridoma formation efficiency (5)
Z10 (18)
CMV
5
2 × 106
SBC-H2O
Iso-osmolar (14)
3.0 kV/cm 3 pulses/15 µs
34–68
X2-16 (19)
CMV
100
1.4 × 106
K6H6/B5
Iso-osmolar (14)
3.0 kV/cm 3 pulses/15 µs
6–18
IH-9 (20)
HTLV-I
3
105
K6H6/B5
Hypo-osmolar 75L3
1.0 kV/cm 3 pulses/15 µs
141–258
WA-04 2B10 (4)
HTLV-I
40
1.5 × 105
H73C11
Hypo-osmolar 100L3
1.25 kV/cm 1 pulse/10 µs
57–283
WA-11 1F5 (4)
HTLV-I
70
1.5 × 106
K6H6/B5
Iso-osmolar 300L3
1.75 kV/cm 3 pulses/15 µs
40–96
JB-04 1D7
HCV
90
106
K6H6/B5
Iso-osmolar 300L3
1.75 kV/cm 3 pulses/15 µs
11–66
JB-16 2D6
HCV
14
1.5 × 105
H73C11
Hypo-osmolar 100L3
1.25 kV/cm 1 pulse/10 µs
194–388
JB-17 1D2
HCV
60
1.5 × 105
K6H6/B5
Hypo-osmolar 100L3
1.25 kV/cm 3 pulses/15 µs
80–200
a
(The number of wells with growth × The range in colony#/well in over 1/2 the wells) / 105 input EBV-activated B-cells.
12/1994: pages 295-307;
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Vox Sanguinis - VOX SANG. 01/1985; 49(3):206-210.
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