CORD-19-Sentences  

CORD-19:0049ba8861864506e1e8559e7815f4de8b03dbed JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
TextSentencer_T1 0-125 Sentence denotes GPI-anchored single chain Fv -an effective way to capture transiently-exposed neutralization epitopes on HIV-1 envelope spike
T89605 0-125 Sentence denotes GPI-anchored single chain Fv -an effective way to capture transiently-exposed neutralization epitopes on HIV-1 envelope spike
TextSentencer_T2 127-135 Sentence denotes Abstract
T72116 127-135 Sentence denotes Abstract
TextSentencer_T3 136-147 Sentence denotes Background:
T77916 136-147 Sentence denotes Background:
TextSentencer_T4 148-264 Sentence denotes Identification of broad neutralization epitopes in HIV-1 envelope spikes is paramount for HIV-1 vaccine development.
T56480 148-264 Sentence denotes Identification of broad neutralization epitopes in HIV-1 envelope spikes is paramount for HIV-1 vaccine development.
TextSentencer_T5 265-475 Sentence denotes A few broad neutralization epitopes identified so far are present on the surface of native HIV-1 envelope spikes whose recognition by antibodies does not depend on conformational changes of the envelope spikes.
T16620 265-475 Sentence denotes A few broad neutralization epitopes identified so far are present on the surface of native HIV-1 envelope spikes whose recognition by antibodies does not depend on conformational changes of the envelope spikes.
TextSentencer_T6 476-602 Sentence denotes However, HIV-1 envelope spikes also contain transiently-exposed neutralization epitopes, which are more difficult to identify.
T16925 476-602 Sentence denotes However, HIV-1 envelope spikes also contain transiently-exposed neutralization epitopes, which are more difficult to identify.
TextSentencer_T7 603-611 Sentence denotes Results:
T84846 603-611 Sentence denotes Results:
TextSentencer_T8 612-814 Sentence denotes In this study, we constructed single chain Fvs (scFvs) derived from seven human monoclonal antibodies and genetically linked them with or without a glycosyl-phosphatidylinositol (GPI) attachment signal.
T6273 612-814 Sentence denotes In this study, we constructed single chain Fvs (scFvs) derived from seven human monoclonal antibodies and genetically linked them with or without a glycosyl-phosphatidylinositol (GPI) attachment signal.
TextSentencer_T9 815-915 Sentence denotes We show that with a GPI attachment signal the scFvs are targeted to lipid rafts of plasma membranes.
T68017 815-915 Sentence denotes We show that with a GPI attachment signal the scFvs are targeted to lipid rafts of plasma membranes.
TextSentencer_T10 916-1079 Sentence denotes In addition, we demonstrate that four of the GPI-anchored scFvs, but not their secreted counterparts, neutralize HIV-1 with various degrees of breadth and potency.
T52103 916-1079 Sentence denotes In addition, we demonstrate that four of the GPI-anchored scFvs, but not their secreted counterparts, neutralize HIV-1 with various degrees of breadth and potency.
TextSentencer_T11 1080-1223 Sentence denotes Among them, GPI-anchored scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 strains tested.
T93008 1080-1223 Sentence denotes Among them, GPI-anchored scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 strains tested.
TextSentencer_T12 1224-1354 Sentence denotes Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart.
T5034 1224-1354 Sentence denotes Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart.
TextSentencer_T13 1355-1636 Sentence denotes Finally, we demonstrate that expression of GPI-anchored scFv (X5) in the lipid raft of plasma membrane of human CD4 + T cells confers longterm resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion, and the infection of HIV-1 captured and transferred by human DCs.
T5505 1355-1636 Sentence denotes Finally, we demonstrate that expression of GPI-anchored scFv (X5) in the lipid raft of plasma membrane of human CD4 + T cells confers longterm resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion, and the infection of HIV-1 captured and transferred by human DCs.
TextSentencer_T14 1637-1649 Sentence denotes Conclusions:
T9421 1637-1649 Sentence denotes Conclusions:
TextSentencer_T15 1650-1859 Sentence denotes Thus GPI-anchored scFv could be used as a general and effective way to identify antibodies that react with transiently-exposed neutralization epitopes in envelope proteins of HIV-1 and other enveloped viruses.
T30378 1650-1859 Sentence denotes Thus GPI-anchored scFv could be used as a general and effective way to identify antibodies that react with transiently-exposed neutralization epitopes in envelope proteins of HIV-1 and other enveloped viruses.
TextSentencer_T16 1860-2025 Sentence denotes The GPI-anchored scFv (X5), because of its breadth and potency, should have a great potential to be developed into anti-viral agent for HIV-1 prevention and therapy.
T62164 1860-2025 Sentence denotes The GPI-anchored scFv (X5), because of its breadth and potency, should have a great potential to be developed into anti-viral agent for HIV-1 prevention and therapy.
TextSentencer_T17 2027-2180 Sentence denotes Human Immunodeficiency Virus type 1 (HIV-1) envelope spike is a trimeric complex consisting of three noncovalently linked heterodimers of gp120 and gp41.
T35204 2027-2180 Sentence denotes Human Immunodeficiency Virus type 1 (HIV-1) envelope spike is a trimeric complex consisting of three noncovalently linked heterodimers of gp120 and gp41.
TextSentencer_T18 2181-2273 Sentence denotes Gp120, an exterior glycoprotein, mediates cell attachment, receptor and co-receptor binding.
T83499 2181-2273 Sentence denotes Gp120, an exterior glycoprotein, mediates cell attachment, receptor and co-receptor binding.
TextSentencer_T19 2274-2406 Sentence denotes Gp41, a transmembrane glycoprotein, mediates viral and cell membrane fusion, which is critical for viral core to enter target cells.
T18115 2274-2406 Sentence denotes Gp41, a transmembrane glycoprotein, mediates viral and cell membrane fusion, which is critical for viral core to enter target cells.
TextSentencer_T20 2407-2479 Sentence denotes Both gp120 and gp41 are derived by cleavage of a common precursor gp160.
T25294 2407-2479 Sentence denotes Both gp120 and gp41 are derived by cleavage of a common precursor gp160.
TextSentencer_T21 2480-2533 Sentence denotes HIV-1 envelope spike also elicits antibody responses.
T57450 2480-2533 Sentence denotes HIV-1 envelope spike also elicits antibody responses.
TextSentencer_T22 2534-2776 Sentence denotes Neutralizing antibodies block viral entry by recognizing epitopes on the envelope spike critical for its attachment, receptor and co-receptor interaction, or fusion and appear to be an important component of a protective immune response [1] .
T18548 2534-2776 Sentence denotes Neutralizing antibodies block viral entry by recognizing epitopes on the envelope spike critical for its attachment, receptor and co-receptor interaction, or fusion and appear to be an important component of a protective immune response [1] .
TextSentencer_T23 2777-2904 Sentence denotes However, antibodies that can neutralize a broad range of primary HIV-1 isolates have been extremely difficult to generate [2] .
T73775 2777-2904 Sentence denotes However, antibodies that can neutralize a broad range of primary HIV-1 isolates have been extremely difficult to generate [2] .
TextSentencer_T24 2905-3211 Sentence denotes Despite more than two decades of effort, only a few broadly neutralizing antibodies (2G12, b12, VRC001, VRC002, VRC003, PG9, PG16, 2F5 and 4E10/Z13) have been identified through screening antibody libraries or memory B cells from HIV-1 infected individuals [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] .
T33366 2905-3211 Sentence denotes Despite more than two decades of effort, only a few broadly neutralizing antibodies (2G12, b12, VRC001, VRC002, VRC003, PG9, PG16, 2F5 and 4E10/Z13) have been identified through screening antibody libraries or memory B cells from HIV-1 infected individuals [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] .
TextSentencer_T25 3212-3328 Sentence denotes Unfortunately, many efforts to elicit such antibody responses by active immunization have not been successful [14] .
T39127 3212-3328 Sentence denotes Unfortunately, many efforts to elicit such antibody responses by active immunization have not been successful [14] .
TextSentencer_T26 3329-3583 Sentence denotes Interestingly, neutralization epitopes recognized by the aforementioned broadly neutralizing antibodies are present on the surface of the native spike and their recognition by the antibodies does not depend on conformational changes of envelope proteins.
T24807 3329-3583 Sentence denotes Interestingly, neutralization epitopes recognized by the aforementioned broadly neutralizing antibodies are present on the surface of the native spike and their recognition by the antibodies does not depend on conformational changes of envelope proteins.
TextSentencer_T27 3584-3898 Sentence denotes Upon interaction with CD4 receptor, a lipid raftassociated protein [15] [16] [17] [18] , on the target cell surface, the native HIV-1 envelope spike goes through extensive conformational changes that allow additional binding to a co-receptor, CXCR4 for T-cell tropic strains or CCR5 for macrophage-tropic isolates.
T43742 3584-3898 Sentence denotes Upon interaction with CD4 receptor, a lipid raftassociated protein [15] [16] [17] [18] , on the target cell surface, the native HIV-1 envelope spike goes through extensive conformational changes that allow additional binding to a co-receptor, CXCR4 for T-cell tropic strains or CCR5 for macrophage-tropic isolates.
TextSentencer_T28 3899-4081 Sentence denotes Co-receptor binding results in further conformational changes and leads to the insertion of the fusion peptide in gp41 into target cell membrane to drive the subsequent fusion event.
T64196 3899-4081 Sentence denotes Co-receptor binding results in further conformational changes and leads to the insertion of the fusion peptide in gp41 into target cell membrane to drive the subsequent fusion event.
TextSentencer_T29 4082-4310 Sentence denotes During these conformational changes epitopes that are hidden from or not totally exposed on the surface of native spike are transiently exposed and become accessible to antibodies specific for these transiently-exposed epitopes.
T55584 4082-4310 Sentence denotes During these conformational changes epitopes that are hidden from or not totally exposed on the surface of native spike are transiently exposed and become accessible to antibodies specific for these transiently-exposed epitopes.
TextSentencer_T30 4311-4375 Sentence denotes Likely, some of these epitopes are also neutralization epitopes.
T29436 4311-4375 Sentence denotes Likely, some of these epitopes are also neutralization epitopes.
TextSentencer_T31 4376-4657 Sentence denotes Based on this assumption, several groups reported using gp120-CD4 or gp120-CD4-CCR5 complex as immunogens to elicit antibodies that react with transiently-exposed neutralization epitopes or as selecting antigens for screening human phage display antibody libraries [19] [20] [21] .
T3071 4376-4657 Sentence denotes Based on this assumption, several groups reported using gp120-CD4 or gp120-CD4-CCR5 complex as immunogens to elicit antibodies that react with transiently-exposed neutralization epitopes or as selecting antigens for screening human phage display antibody libraries [19] [20] [21] .
TextSentencer_T32 4658-4880 Sentence denotes It was hypothesized that in these complexes HIV-1 envelope may stabilize some of the transiently-exposed epitopes so that antibodies present in the libraries that recognized these stabilized epitopes can be selected [22] .
T34235 4658-4880 Sentence denotes It was hypothesized that in these complexes HIV-1 envelope may stabilize some of the transiently-exposed epitopes so that antibodies present in the libraries that recognized these stabilized epitopes can be selected [22] .
TextSentencer_T33 4881-5030 Sentence denotes One notable example was the identification of a CD4inducible antibody X5 in a phage display Fab antibody library with a gp120-CD4-CCR5 complex [21] .
T87381 4881-5030 Sentence denotes One notable example was the identification of a CD4inducible antibody X5 in a phage display Fab antibody library with a gp120-CD4-CCR5 complex [21] .
TextSentencer_T34 5031-5369 Sentence denotes Previously, we unexpectedly found that by genetically linking the scFv of an anti-HIV-1 human antibody (TG15) to the transmembrane domain of subunit one of the type 1 interferon receptor, the cell-surface expressed scFv, but not its secretory form, we markedly inhibited HIV-1 entry and HIV-1 envelope-mediated cell-cell fusion [23, 24] .
T2438 5031-5369 Sentence denotes Previously, we unexpectedly found that by genetically linking the scFv of an anti-HIV-1 human antibody (TG15) to the transmembrane domain of subunit one of the type 1 interferon receptor, the cell-surface expressed scFv, but not its secretory form, we markedly inhibited HIV-1 entry and HIV-1 envelope-mediated cell-cell fusion [23, 24] .
TextSentencer_T35 5370-5523 Sentence denotes The antibody recognizes the cluster II determinant (amino acid residues 644-663) which resides within the second heptad repeat (HR2) of HIV-1 gp41 [25] .
T93237 5370-5523 Sentence denotes The antibody recognizes the cluster II determinant (amino acid residues 644-663) which resides within the second heptad repeat (HR2) of HIV-1 gp41 [25] .
TextSentencer_T36 5524-5712 Sentence denotes HIV-1 gp41 mediated fusion is triggered by interaction between the second and the first heptad repeats, which converts a prehairpin gp41 trimer into a fusogenic three-hairpin bundle [26] .
T45467 5524-5712 Sentence denotes HIV-1 gp41 mediated fusion is triggered by interaction between the second and the first heptad repeats, which converts a prehairpin gp41 trimer into a fusogenic three-hairpin bundle [26] .
TextSentencer_T37 5713-6044 Sentence denotes Similarly, it was reported that expressing a peptide derived from the HR2 domain on the surface of HIV-1-susceptible cells exhibits greater inhibitory effect on HIV-1 [27] and such an inhibition is achieved by capturing a gp41 fusion intermediate by the cell-surface expressed peptide prior to viral and cell membrane fusion [28] .
T1586 5713-6044 Sentence denotes Similarly, it was reported that expressing a peptide derived from the HR2 domain on the surface of HIV-1-susceptible cells exhibits greater inhibitory effect on HIV-1 [27] and such an inhibition is achieved by capturing a gp41 fusion intermediate by the cell-surface expressed peptide prior to viral and cell membrane fusion [28] .
TextSentencer_T38 6045-6230 Sentence denotes Thus, it is clear that the cellsurface expressed scFv or peptide that recognizes or is derived from the HR2 domain can capture transientlyexposed epitopes in entry fusion intermediates.
T35627 6045-6230 Sentence denotes Thus, it is clear that the cellsurface expressed scFv or peptide that recognizes or is derived from the HR2 domain can capture transientlyexposed epitopes in entry fusion intermediates.
TextSentencer_T39 6231-6413 Sentence denotes However, it is not clear whether transiently-exposed epitopes on HIV-1 envelope spikes other than that resides in the HR2 domain can also be captured by cell-surface expressed scFvs.
T91139 6231-6413 Sentence denotes However, it is not clear whether transiently-exposed epitopes on HIV-1 envelope spikes other than that resides in the HR2 domain can also be captured by cell-surface expressed scFvs.
TextSentencer_T40 6414-6589 Sentence denotes In nature, over 200 cell surface proteins with various functions are anchored to the plasma membrane by a covalently attached glycosyl-phosphatidylinositol (GPI) anchor [29] .
T28095 6414-6589 Sentence denotes In nature, over 200 cell surface proteins with various functions are anchored to the plasma membrane by a covalently attached glycosyl-phosphatidylinositol (GPI) anchor [29] .
TextSentencer_T41 6590-6674 Sentence denotes Many GPI-anchored proteins are targeted into the lipid rafts of the plasma membrane.
T19788 6590-6674 Sentence denotes Many GPI-anchored proteins are targeted into the lipid rafts of the plasma membrane.
TextSentencer_T42 6675-6785 Sentence denotes These specialized dynamic micro-domains are rich in cholesterol, sphingolipids and glycerophospholipids [30] .
T50101 6675-6785 Sentence denotes These specialized dynamic micro-domains are rich in cholesterol, sphingolipids and glycerophospholipids [30] .
TextSentencer_T43 6786-6856 Sentence denotes The lipid raft has been known to be a gateway for HIV-1 budding [31] .
T82517 6786-6856 Sentence denotes The lipid raft has been known to be a gateway for HIV-1 budding [31] .
TextSentencer_T44 6857-6985 Sentence denotes Furthermore, involvement of lipid rafts in HIV-1 entry into T cells and macrophages has also been proposed [15, [31] [32] [33] .
T60882 6857-6985 Sentence denotes Furthermore, involvement of lipid rafts in HIV-1 entry into T cells and macrophages has also been proposed [15, [31] [32] [33] .
TextSentencer_T45 6986-7289 Sentence denotes We therefore hypothesized that if one can express antibodies that react with transiently-exposed neutralization epitopes in a GPI anchored form and a GPI anchor can target these antibodies into the lipid rafts of plasma membranes of HIV-1-susceptible cells, these antibodies should neutralize infection.
T81377 6986-7289 Sentence denotes We therefore hypothesized that if one can express antibodies that react with transiently-exposed neutralization epitopes in a GPI anchored form and a GPI anchor can target these antibodies into the lipid rafts of plasma membranes of HIV-1-susceptible cells, these antibodies should neutralize infection.
TextSentencer_T46 7290-7573 Sentence denotes If correct, we predict that when the HIV-1 native spike interacts with the CD4 receptor, triggering a series of conformational changes, the transiently-exposed neutralization epitopes will be captured by GPI-anchored antibodies residing in the same lipid raft of the plasma membrane.
T6542 7290-7573 Sentence denotes If correct, we predict that when the HIV-1 native spike interacts with the CD4 receptor, triggering a series of conformational changes, the transiently-exposed neutralization epitopes will be captured by GPI-anchored antibodies residing in the same lipid raft of the plasma membrane.
TextSentencer_T47 7574-7732 Sentence denotes To test this hypothesis in this study, we constructed scFvs derived from seven different human monoclonal antibodies AB31, AB32, TG15, 4E10, 48d, X5 and AB65.
T94035 7574-7732 Sentence denotes To test this hypothesis in this study, we constructed scFvs derived from seven different human monoclonal antibodies AB31, AB32, TG15, 4E10, 48d, X5 and AB65.
TextSentencer_T48 7733-7837 Sentence denotes AB65 recognizes the influenza hemagglutinin used here as negative control (Zhou, et al. data not shown).
T83828 7733-7837 Sentence denotes AB65 recognizes the influenza hemagglutinin used here as negative control (Zhou, et al. data not shown).
TextSentencer_T49 7838-7881 Sentence denotes AB31 and AB32 are high affinity antibodies.
T39874 7838-7881 Sentence denotes AB31 and AB32 are high affinity antibodies.
TextSentencer_T50 7882-8009 Sentence denotes AB31 recognizes cluster III determinant of gp41 and AB32 interacts with gp120, but its epitope is not well characterized [34] .
T44223 7882-8009 Sentence denotes AB31 recognizes cluster III determinant of gp41 and AB32 interacts with gp120, but its epitope is not well characterized [34] .
TextSentencer_T51 8010-8166 Sentence denotes Antibody (TG15) recognizes the cluster II determinant (amino acid residues 644-663) which resides within the second heptad repeat (HR2) of HIV-1 gp41 [23] .
T6557 8010-8166 Sentence denotes Antibody (TG15) recognizes the cluster II determinant (amino acid residues 644-663) which resides within the second heptad repeat (HR2) of HIV-1 gp41 [23] .
TextSentencer_T52 8167-8351 Sentence denotes Antibodies 48d and X5 recognize distinct, but partially overlapped CD4 induced epitopes that are located close to both co-receptor-binding and CD4-binding sites of gp120 [21, 35, 36] .
T21069 8167-8351 Sentence denotes Antibodies 48d and X5 recognize distinct, but partially overlapped CD4 induced epitopes that are located close to both co-receptor-binding and CD4-binding sites of gp120 [21, 35, 36] .
TextSentencer_T53 8352-8481 Sentence denotes Antibody 4E10 that recognizes a linear epitope residing in the membrane proximate region of gp41 is a neutralizing antibody [7] .
T97318 8352-8481 Sentence denotes Antibody 4E10 that recognizes a linear epitope residing in the membrane proximate region of gp41 is a neutralizing antibody [7] .
TextSentencer_T54 8482-8671 Sentence denotes Here, we show that by genetically linking the scFvs with a GPI attachment signal derived from decay accelerating factor (DAF) [37] the scFvs are targeted to lipid rafts of plasma membranes.
T5044 8482-8671 Sentence denotes Here, we show that by genetically linking the scFvs with a GPI attachment signal derived from decay accelerating factor (DAF) [37] the scFvs are targeted to lipid rafts of plasma membranes.
TextSentencer_T55 8672-8866 Sentence denotes In addition, we demonstrate that the four of these GPIanchored scFvs (X5, 48d, AB32 and TG15), but not their secretory counterparts, neutralize HIV-1 with various degrees of breadth and potency.
T26011 8672-8866 Sentence denotes In addition, we demonstrate that the four of these GPIanchored scFvs (X5, 48d, AB32 and TG15), but not their secretory counterparts, neutralize HIV-1 with various degrees of breadth and potency.
TextSentencer_T56 8867-9009 Sentence denotes Among them, GPIanchored scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 strains tested.
T72578 8867-9009 Sentence denotes Among them, GPIanchored scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 strains tested.
TextSentencer_T57 9010-9140 Sentence denotes Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart.
T37689 9010-9140 Sentence denotes Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart.
TextSentencer_T58 9141-9422 Sentence denotes Finally, we demonstrate that expression of GPI-anchored scFv (X5) in the lipid raft of plasma membrane of human CD4 + T cells confers long-term resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion and the infection of HIV-1 captured and transferred by human DCs.
T42625 9141-9422 Sentence denotes Finally, we demonstrate that expression of GPI-anchored scFv (X5) in the lipid raft of plasma membrane of human CD4 + T cells confers long-term resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion and the infection of HIV-1 captured and transferred by human DCs.
TextSentencer_T59 9423-9567 Sentence denotes Thus, we conclude that GPI-anchored scFv is an effective way to capture transiently-exposed neutralization epitopes in the HIV-1 envelope spike.
T36949 9423-9567 Sentence denotes Thus, we conclude that GPI-anchored scFv is an effective way to capture transiently-exposed neutralization epitopes in the HIV-1 envelope spike.
TextSentencer_T60 9568-9644 Sentence denotes Expression of scFv in the lipid raft of plasma membrane through a GPI anchor
T29328 9568-9644 Sentence denotes Expression of scFv in the lipid raft of plasma membrane through a GPI anchor
TextSentencer_T61 9645-9968 Sentence denotes To generate GPI-anchored and secretory scFvs, the sequences encoding scFvs derived from seven different human antibodies AB31, AB32, TG15, 4E10, 48d, X5 and AB65 were genetically linked with the sequence encoding a his-tagged IgG3 hinge region and with or without the sequence encoding a GPI attachment signal of DAF [37] .
T46109 9645-9968 Sentence denotes To generate GPI-anchored and secretory scFvs, the sequences encoding scFvs derived from seven different human antibodies AB31, AB32, TG15, 4E10, 48d, X5 and AB65 were genetically linked with the sequence encoding a his-tagged IgG3 hinge region and with or without the sequence encoding a GPI attachment signal of DAF [37] .
TextSentencer_T62 9969-10118 Sentence denotes The fusion genes scFv/IgG3 hinge/his-tag/ DAF and scFv/IgG3 hinge/his-tag were inserted into a third generation lentiviral vector pRRL ( Figure 1A ).
T13186 9969-10118 Sentence denotes The fusion genes scFv/IgG3 hinge/his-tag/ DAF and scFv/IgG3 hinge/his-tag were inserted into a third generation lentiviral vector pRRL ( Figure 1A ).
TextSentencer_T63 10119-10280 Sentence denotes The recombinant viruses were then generated as described before [38] and used to transduce TZM.bl cells and human CD4 + T cells CEMss and CEMss-CCR5 (see below).
T7372 10119-10280 Sentence denotes The recombinant viruses were then generated as described before [38] and used to transduce TZM.bl cells and human CD4 + T cells CEMss and CEMss-CCR5 (see below).
TextSentencer_T64 10281-10396 Sentence denotes The expression of transgenes and localization of transgene products in the transduced cells were carefully studied.
T31921 10281-10396 Sentence denotes The expression of transgenes and localization of transgene products in the transduced cells were carefully studied.
TextSentencer_T65 10397-10611 Sentence denotes Figure 1B shows the expression of scFvs/hinge/his-tag/ DAF and scFvs/hinge/his-tag in cell lysates and culture supernatants of transduced TZM.bl cells by western blot using anti-his-tag and anti-tubulin antibodies.
T77025 10397-10611 Sentence denotes Figure 1B shows the expression of scFvs/hinge/his-tag/ DAF and scFvs/hinge/his-tag in cell lysates and culture supernatants of transduced TZM.bl cells by western blot using anti-his-tag and anti-tubulin antibodies.
TextSentencer_T66 10612-10778 Sentence denotes As expected, without a GPI attachment signal, all scFvs were detected in both culture supernatants and cell lysates with a majority in supernatants (the right panel).
T66147 10612-10778 Sentence denotes As expected, without a GPI attachment signal, all scFvs were detected in both culture supernatants and cell lysates with a majority in supernatants (the right panel).
TextSentencer_T67 10779-10916 Sentence denotes By contrast, all scFvs with a GPI attachment signal were only detected in cell lysates, but not in culture supernatants (the left panel).
T6868 10779-10916 Sentence denotes By contrast, all scFvs with a GPI attachment signal were only detected in cell lysates, but not in culture supernatants (the left panel).
TextSentencer_T68 10917-11011 Sentence denotes These data indicate that inclusion of a GPI attachment signal prevents secretion of the scFvs.
T61609 10917-11011 Sentence denotes These data indicate that inclusion of a GPI attachment signal prevents secretion of the scFvs.
TextSentencer_T69 11012-11305 Sentence denotes To determine if scFvs/hinge/his-tag/DAF were expressed on the cell surface through a GPI anchor, scFv/hinge/his-tag/DAF-transduced TZM.bl cells were treated with or without phosphatidylinositol-specific phospholipase C (PI-PLC) and stained with anti-his-tag antibody followed by FACS analysis.
T13383 11012-11305 Sentence denotes To determine if scFvs/hinge/his-tag/DAF were expressed on the cell surface through a GPI anchor, scFv/hinge/his-tag/DAF-transduced TZM.bl cells were treated with or without phosphatidylinositol-specific phospholipase C (PI-PLC) and stained with anti-his-tag antibody followed by FACS analysis.
TextSentencer_T70 11306-11522 Sentence denotes As a control, cells transduced with previously reported m-scFv (TG15), a cell-surface expressed scFv (TG15) with a conventional transmembrane domain [23] went through the same PI-PLC treatment and staining processes.
T10448 11306-11522 Sentence denotes As a control, cells transduced with previously reported m-scFv (TG15), a cell-surface expressed scFv (TG15) with a conventional transmembrane domain [23] went through the same PI-PLC treatment and staining processes.
TextSentencer_T71 11523-11714 Sentence denotes Figure 1C shows that all scFv/hinge/his-tag/DAFs express highly on cell surface (about 10-fold higher than that of m-scFv) and the expression were substantially reduced with PI-PLC treatment.
T46385 11523-11714 Sentence denotes Figure 1C shows that all scFv/hinge/his-tag/DAFs express highly on cell surface (about 10-fold higher than that of m-scFv) and the expression were substantially reduced with PI-PLC treatment.
TextSentencer_T72 11715-11924 Sentence denotes In contrast, no reduction in cell surface expression of scFv was observed in m-scFv-transduced cells, indicating that the expression of scFv/hinge/histag/DAF on the cell surface is indeed through a GPI anchor.
T82787 11715-11924 Sentence denotes In contrast, no reduction in cell surface expression of scFv was observed in m-scFv-transduced cells, indicating that the expression of scFv/hinge/histag/DAF on the cell surface is indeed through a GPI anchor.
TextSentencer_T73 11925-12088 Sentence denotes In addition, cell surface expression of GPIanchored scFv (4E10) along with GPI-anchored scFvs (AB65 and X5) was also analyzed by immune staining and FACS analysis.
T42026 11925-12088 Sentence denotes In addition, cell surface expression of GPIanchored scFv (4E10) along with GPI-anchored scFvs (AB65 and X5) was also analyzed by immune staining and FACS analysis.
TextSentencer_T74 12089-12223 Sentence denotes Additional File 1 shows that cell surface expression of GPI-anchored scFv (4E10) is similar to those GPI-anchored scFvs (AB65 and X5).
T46489 12089-12223 Sentence denotes Additional File 1 shows that cell surface expression of GPI-anchored scFv (4E10) is similar to those GPI-anchored scFvs (AB65 and X5).
TextSentencer_T75 12224-12374 Sentence denotes Thus, for the sake of simplicity in the remaining text we will refer the scFv/hinge/his-tag/DAF as GPI-scFv and scFv/hinge/ his-tag as secretory scFv.
T20991 12224-12374 Sentence denotes Thus, for the sake of simplicity in the remaining text we will refer the scFv/hinge/his-tag/DAF as GPI-scFv and scFv/hinge/ his-tag as secretory scFv.
TextSentencer_T76 12375-12578 Sentence denotes To determine if GPI-scFvs are located in the lipid rafts of plasma membranes, mock-and GPI-scFv (AB65 and X5)-transduced TZM.bl cells were seeded into wells of cover slip chambers and cultured overnight.
T87816 12375-12578 Sentence denotes To determine if GPI-scFvs are located in the lipid rafts of plasma membranes, mock-and GPI-scFv (AB65 and X5)-transduced TZM.bl cells were seeded into wells of cover slip chambers and cultured overnight.
TextSentencer_T77 12579-12802 Sentence denotes Cells were then fixed with 4% formaldehyde and co-stained with 1) mouse anti-his-tag antibody followed by Alexa 488conjugated goat anti-mouse IgG antibody; 2) Alexa 555conjugated cholera toxin subunit B (CtxB); and 3) DAPI.
T11671 12579-12802 Sentence denotes Cells were then fixed with 4% formaldehyde and co-stained with 1) mouse anti-his-tag antibody followed by Alexa 488conjugated goat anti-mouse IgG antibody; 2) Alexa 555conjugated cholera toxin subunit B (CtxB); and 3) DAPI.
TextSentencer_T78 12803-12869 Sentence denotes CtxB interacts with GM1 (a lipid raft marker) on the cell surface.
T15298 12803-12869 Sentence denotes CtxB interacts with GM1 (a lipid raft marker) on the cell surface.
TextSentencer_T79 12870-13034 Sentence denotes Figure 2 shows that both GPI-scFvs (AB65 and X5) are co-localized with GM1 on cell surface, implying that they are located in the lipid raft of the plasma membrane.
T34284 12870-13034 Sentence denotes Figure 2 shows that both GPI-scFvs (AB65 and X5) are co-localized with GM1 on cell surface, implying that they are located in the lipid raft of the plasma membrane.
TextSentencer_T80 13035-13112 Sentence denotes GPI-scFv (X5) exhibits remarkable degree of breadth and potency against HIV-1
T43773 13035-13112 Sentence denotes GPI-scFv (X5) exhibits remarkable degree of breadth and potency against HIV-1
TextSentencer_T81 13113-13493 Sentence denotes Next, we compared CD4, CCR5 and CXCR4 expression in the secretory and GPI-scFv-transduced TZM.bl cells and found that there is no significant difference in their expression compared to mock-transduced TZM.bl cells, suggesting that the expression of transgenes does not alter the expression of the receptor and the coreceptors for HIV-1 in the transduced cells (Additional File 2).
T19855 13113-13493 Sentence denotes Next, we compared CD4, CCR5 and CXCR4 expression in the secretory and GPI-scFv-transduced TZM.bl cells and found that there is no significant difference in their expression compared to mock-transduced TZM.bl cells, suggesting that the expression of transgenes does not alter the expression of the receptor and the coreceptors for HIV-1 in the transduced cells (Additional File 2).
TextSentencer_T82 13494-13604 Sentence denotes Neither did we find that the expression of the transgenes alters the cell growth (Zhou et al. data not shown).
T6336 13494-13604 Sentence denotes Neither did we find that the expression of the transgenes alters the cell growth (Zhou et al. data not shown).
TextSentencer_T83 13605-13865 Sentence denotes To test neutralization activity of the secretory versus the GPI-scFvs against HIV-1, an eleven multiclade HIV-1 pseudotype panel and a retroviral envelope 10A1 pseudotype were used to infect transduced TZM.bl cells in a single-round infection experiment [23] .
T97028 13605-13865 Sentence denotes To test neutralization activity of the secretory versus the GPI-scFvs against HIV-1, an eleven multiclade HIV-1 pseudotype panel and a retroviral envelope 10A1 pseudotype were used to infect transduced TZM.bl cells in a single-round infection experiment [23] .
TextSentencer_T84 13866-13997 Sentence denotes The retroviral envelope 10A1 recognizes either Ram-1 or Glvr-1 as a receptor for cell entry [39] and used here as negative control.
T89427 13866-13997 Sentence denotes The retroviral envelope 10A1 recognizes either Ram-1 or Glvr-1 as a receptor for cell entry [39] and used here as negative control.
TextSentencer_T85 13998-14199 Sentence denotes The eleven HIV-1 pseudotypes consist of HIV-1 envelopes derived from clade A (Q168), clade B (HxBc2, JF-RL, ADA, AD8, Yu2 and consensus B), clade B' (CNE11), clade C (Mj4 and CNE17) and clade E (CNE8).
T54661 13998-14199 Sentence denotes The eleven HIV-1 pseudotypes consist of HIV-1 envelopes derived from clade A (Q168), clade B (HxBc2, JF-RL, ADA, AD8, Yu2 and consensus B), clade B' (CNE11), clade C (Mj4 and CNE17) and clade E (CNE8).
TextSentencer_T86 14200-14363 Sentence denotes Figure 3 shows mean and standard deviation of relative luciferase activity (RLA) in mock-, secretory and GPI-scFv-transduced cells infected with these pseudotypes.
T22674 14200-14363 Sentence denotes Figure 3 shows mean and standard deviation of relative luciferase activity (RLA) in mock-, secretory and GPI-scFv-transduced cells infected with these pseudotypes.
TextSentencer_T87 14364-14560 Sentence denotes Compared to mock-transduced cells, cells transduced with all secretory and GPI-anchored scFvs did not show significant neutralization activity against 10A1 pseudotypes control ( Figure 3A and 3B).
T52673 14364-14560 Sentence denotes Compared to mock-transduced cells, cells transduced with all secretory and GPI-anchored scFvs did not show significant neutralization activity against 10A1 pseudotypes control ( Figure 3A and 3B).
TextSentencer_T88 14561-14763 Sentence denotes Compared to mock-transduced cells, cells transduced with secretory scFvs (AB65, AB31, AB32, TG15, and 48d) did not show significant neutralization activity against any of these HIV-1 pseudotypes tested.
T41945 14561-14763 Sentence denotes Compared to mock-transduced cells, cells transduced with secretory scFvs (AB65, AB31, AB32, TG15, and 48d) did not show significant neutralization activity against any of these HIV-1 pseudotypes tested.
TextSentencer_T89 14764-14912 Sentence denotes Cells transduced with secretory scFv (X5) showed low degree of neutralization activity against 3 of 11 HIV-1 pseudotypes (ADA, Consensus B and Mj4).
T66747 14764-14912 Sentence denotes Cells transduced with secretory scFv (X5) showed low degree of neutralization activity against 3 of 11 HIV-1 pseudotypes (ADA, Consensus B and Mj4).
TextSentencer_T90 14913-15072 Sentence denotes In contrast, cells transduced with secretory scFv (4E10) exhibited more than 50% neutralization activity against all 11 HIV-1 pseudotypes tested ( Figure 3B ).
T44432 14913-15072 Sentence denotes In contrast, cells transduced with secretory scFv (4E10) exhibited more than 50% neutralization activity against all 11 HIV-1 pseudotypes tested ( Figure 3B ).
TextSentencer_T91 15073-15197 Sentence denotes Compared to mock-transduced cells, cells Figure 1 Expression of secretory and GPI-anchored scFvs in transduced TZM.bl cells.
T53891 15073-15197 Sentence denotes Compared to mock-transduced cells, cells Figure 1 Expression of secretory and GPI-anchored scFvs in transduced TZM.bl cells.
TextSentencer_T92 15198-15200 Sentence denotes A.
T47300 15198-15200 Sentence denotes A.
TextSentencer_T93 15201-15301 Sentence denotes Schematic diagram of the lentiviral vectors pRRL-scFv/hinge/his-tag/DAF and pRRL-scFv/hinge/his-tag.
T11536 15201-15301 Sentence denotes Schematic diagram of the lentiviral vectors pRRL-scFv/hinge/his-tag/DAF and pRRL-scFv/hinge/his-tag.
TextSentencer_T94 15302-15557 Sentence denotes Single chain Fvs (scFvs) were derived from seven human monoclonal antibodies AB31, AB32, TG15, 48d, X5 and AB65; hinge: a human IgG3 hinge region; his-tag: a 6 histidine residue tag; DAF: the C-terminal 34 amino acid residues of decay accelerating factor.
T39705 15302-15557 Sentence denotes Single chain Fvs (scFvs) were derived from seven human monoclonal antibodies AB31, AB32, TG15, 48d, X5 and AB65; hinge: a human IgG3 hinge region; his-tag: a 6 histidine residue tag; DAF: the C-terminal 34 amino acid residues of decay accelerating factor.
TextSentencer_T95 15558-15560 Sentence denotes B.
T15990 15558-15560 Sentence denotes B.
TextSentencer_T96 15561-15750 Sentence denotes Western blot analysis of expression of scFvs (AB31, AB32, TG15, 48d, X5 and AB65) in TZM.bl cells transduced with lentiviral vectors pRRL-scFv/hinge/his-tag/DAF and pRRL-scFv/hinge/his-tag.
T33758 15561-15750 Sentence denotes Western blot analysis of expression of scFvs (AB31, AB32, TG15, 48d, X5 and AB65) in TZM.bl cells transduced with lentiviral vectors pRRL-scFv/hinge/his-tag/DAF and pRRL-scFv/hinge/his-tag.
TextSentencer_T97 15751-15837 Sentence denotes GPI-scFv: GPI-anchored scFv; Sec-scFv: secretory scFv; anti-his: antihis-tag antibody.
T76627 15751-15837 Sentence denotes GPI-scFv: GPI-anchored scFv; Sec-scFv: secretory scFv; anti-his: antihis-tag antibody.
TextSentencer_T98 15838-15840 Sentence denotes C.
T90230 15838-15840 Sentence denotes C.
TextSentencer_T99 15841-16051 Sentence denotes FACS analysis of cell surface expression of scFv/hinge/histag/DAF in mock-, scFvs (AB31, AB32, TG15, 48d, X5 and AB65)/ hinge/histag/DAF-or m-scFv(TG15)-transduced TZM.bl cells with or without PI-PLC treatment.
T76618 15841-16051 Sentence denotes FACS analysis of cell surface expression of scFv/hinge/histag/DAF in mock-, scFvs (AB31, AB32, TG15, 48d, X5 and AB65)/ hinge/histag/DAF-or m-scFv(TG15)-transduced TZM.bl cells with or without PI-PLC treatment.
TextSentencer_T100 16052-16062 Sentence denotes Wen et al.
T11452 16052-16062 Sentence denotes Wen et al.
TextSentencer_T101 16063-16242 Sentence denotes Retrovirology 2010, 7:79 http://www.retrovirology.com/content/7/1/79 transduced with GPI-scFvs show various degree of potency and breadth against HIV-1 pseudotypes ( Figure 3A ).
T7082 16063-16242 Sentence denotes Retrovirology 2010, 7:79 http://www.retrovirology.com/content/7/1/79 transduced with GPI-scFvs show various degree of potency and breadth against HIV-1 pseudotypes ( Figure 3A ).
TextSentencer_T102 16243-16423 Sentence denotes Like cells transduced with GPI-scFvs (AB65) control, cells transduced with GPI-scFvs (AB31) did not show neutralization breadth and potency against any of these pseudotypes tested.
T43259 16243-16423 Sentence denotes Like cells transduced with GPI-scFvs (AB65) control, cells transduced with GPI-scFvs (AB31) did not show neutralization breadth and potency against any of these pseudotypes tested.
TextSentencer_T103 16424-16551 Sentence denotes Cells transduced with GPI-scFv (AB32) neutralized 2 of 11 HIV-1 pseudotypes (JR-FL and Consensus B) with low degree of potency.
T1548 16424-16551 Sentence denotes Cells transduced with GPI-scFv (AB32) neutralized 2 of 11 HIV-1 pseudotypes (JR-FL and Consensus B) with low degree of potency.
TextSentencer_T104 16552-16738 Sentence denotes Cells transduced with GPI-scFv (TG15) neutralized 8 of 11 HIV-1 pseudoviruses expressing envelopes derived from clades A, B and B' with various degree of potency, but not clades C and E.
T17487 16552-16738 Sentence denotes Cells transduced with GPI-scFv (TG15) neutralized 8 of 11 HIV-1 pseudoviruses expressing envelopes derived from clades A, B and B' with various degree of potency, but not clades C and E.
TextSentencer_T105 16739-16936 Sentence denotes Cells transduced with GPI-scFv (4E10) neutralized all 11 HIV-1 pseudotypes with increased potency (more than 90% neutralization activity) as compared to cells transduced with secretory scFv (4E10).
T70239 16739-16936 Sentence denotes Cells transduced with GPI-scFv (4E10) neutralized all 11 HIV-1 pseudotypes with increased potency (more than 90% neutralization activity) as compared to cells transduced with secretory scFv (4E10).
TextSentencer_T106 16937-17178 Sentence denotes Cells transduced with GPI-scFvs (48d) neutralized all 11 HIV-1 pseudotypes with great degree of potency against HIV-1 pseudotypes expressing envelopes derived from clades A, B, B' and E, but less potent against envelope derived from clade C.
T10116 16937-17178 Sentence denotes Cells transduced with GPI-scFvs (48d) neutralized all 11 HIV-1 pseudotypes with great degree of potency against HIV-1 pseudotypes expressing envelopes derived from clades A, B, B' and E, but less potent against envelope derived from clade C.
TextSentencer_T107 17179-17298 Sentence denotes Strikingly, cells transduced with GPI-scFv (X5) neutralized all 11 HIV-1 pseudotypes with remarkable degree of potency.
T54193 17179-17298 Sentence denotes Strikingly, cells transduced with GPI-scFv (X5) neutralized all 11 HIV-1 pseudotypes with remarkable degree of potency.
TextSentencer_T108 17299-17469 Sentence denotes We next tested neutralization activity of the secretory versus the GPI-scFvs against 6 replication competent HIV-1 strains including two clinical isolates (quasispecies).
T4244 17299-17469 Sentence denotes We next tested neutralization activity of the secretory versus the GPI-scFvs against 6 replication competent HIV-1 strains including two clinical isolates (quasispecies).
TextSentencer_T109 17470-17611 Sentence denotes Figure 3C and 3D show mean and standard deviation of RLA in mock-, secretory and GPI-scFv-transduced cells infected with these HIV-1 strains.
T45796 17470-17611 Sentence denotes Figure 3C and 3D show mean and standard deviation of RLA in mock-, secretory and GPI-scFv-transduced cells infected with these HIV-1 strains.
TextSentencer_T110 17612-17794 Sentence denotes Compared to mock-transduced cells, cells transduced with all secretory scFvs did not show significant neutralization activity against any of these HIV-1 strains tested ( Figure 3D ).
T55497 17612-17794 Sentence denotes Compared to mock-transduced cells, cells transduced with all secretory scFvs did not show significant neutralization activity against any of these HIV-1 strains tested ( Figure 3D ).
TextSentencer_T111 17795-17901 Sentence denotes In contrast, cells transduced with the GPI-scFvs show various degree of breadth and potency ( Figure 3C ).
T20330 17795-17901 Sentence denotes In contrast, cells transduced with the GPI-scFvs show various degree of breadth and potency ( Figure 3C ).
TextSentencer_T112 17902-18045 Sentence denotes Like cells transduced with GPI-scFv (AB65) control, cells transduced with GPI-scFv (AB31) did not neutralize any of these HIV-1 strains tested.
T30556 17902-18045 Sentence denotes Like cells transduced with GPI-scFv (AB65) control, cells transduced with GPI-scFv (AB31) did not neutralize any of these HIV-1 strains tested.
TextSentencer_T113 18046-18353 Sentence denotes Cells transduced with GPI-scFv (AB32 and TG15) It was previously showed that the scFv (X5) neutralizes HIV-1 better than the Fab and the whole IgG [40] and the binding and neutralizing capability of scFv (X5) can be greatly enhanced by adding soluble extracellular domains of human CD4 (sCD4) [21, 41, 42] .
T42740 18046-18353 Sentence denotes Cells transduced with GPI-scFv (AB32 and TG15) It was previously showed that the scFv (X5) neutralizes HIV-1 better than the Fab and the whole IgG [40] and the binding and neutralizing capability of scFv (X5) can be greatly enhanced by adding soluble extracellular domains of human CD4 (sCD4) [21, 41, 42] .
TextSentencer_T114 18354-18467 Sentence denotes We therefore produced and purified soluble CD4 using the drosophila S2 expression system (see Additional File 3).
T58325 18354-18467 Sentence denotes We therefore produced and purified soluble CD4 using the drosophila S2 expression system (see Additional File 3).
TextSentencer_T115 18468-18762 Sentence denotes We then tested the effect of sCD4 doses on HIV-1 infection (Bru-3, Bru-Yu2 and Mj4) and found that at 1 μg/ml or higher a concentration-dependent inhibition of HIV-1 infection by sCD4 was observed; while below 1 μg/ml no significant inhibition by sCD4 was observed (Zhou et al. data not shown).
T52806 18468-18762 Sentence denotes We then tested the effect of sCD4 doses on HIV-1 infection (Bru-3, Bru-Yu2 and Mj4) and found that at 1 μg/ml or higher a concentration-dependent inhibition of HIV-1 infection by sCD4 was observed; while below 1 μg/ml no significant inhibition by sCD4 was observed (Zhou et al. data not shown).
TextSentencer_T116 18763-18882 Sentence denotes Thus, we chose sCD4 at the concentration of 0.3 μg/ml in the subsequent post-CD4 experiments as described before [43] .
T67709 18763-18882 Sentence denotes Thus, we chose sCD4 at the concentration of 0.3 μg/ml in the subsequent post-CD4 experiments as described before [43] .
TextSentencer_T117 18883-19083 Sentence denotes Figure 4 shows mean and standard deviation of RLA in mock-, secretory and GPI-scFv-transduced TZM.bl cells infected with or without HIV-1 Bru-3 or Bru-Yu2 that were pre-incubated with or without sCD4.
T2864 18883-19083 Sentence denotes Figure 4 shows mean and standard deviation of RLA in mock-, secretory and GPI-scFv-transduced TZM.bl cells infected with or without HIV-1 Bru-3 or Bru-Yu2 that were pre-incubated with or without sCD4.
TextSentencer_T118 19084-19453 Sentence denotes Preincubation of 400 and 4,000 TCID 50 of these two HIV-1 strains with sCD4 greatly enhances inhibition in cells transduced with secretory scFv (X5); while complete inhibition was observed in GPI-scFv (X5)-transduced cells infected with 400 and 4,000 TCID 50 of these two HIV-1 strains, regardless whether the viruses were preincubated with sCD4 or not ( Figure 4A -D).
T74815 19084-19453 Sentence denotes Preincubation of 400 and 4,000 TCID 50 of these two HIV-1 strains with sCD4 greatly enhances inhibition in cells transduced with secretory scFv (X5); while complete inhibition was observed in GPI-scFv (X5)-transduced cells infected with 400 and 4,000 TCID 50 of these two HIV-1 strains, regardless whether the viruses were preincubated with sCD4 or not ( Figure 4A -D).
TextSentencer_T119 19454-19655 Sentence denotes Thus, these results clearly show that while sCD4 enhances inhibition by secretory scFv (X5); GPI-scFv (X5) exhibits the greatest potency of inhibition, which is totally independent of addition of sCD4.
T21796 19454-19655 Sentence denotes Thus, these results clearly show that while sCD4 enhances inhibition by secretory scFv (X5); GPI-scFv (X5) exhibits the greatest potency of inhibition, which is totally independent of addition of sCD4.
TextSentencer_T120 19656-19837 Sentence denotes GPI-scFv (X5) confers long-term resistance to HIV-1 in human CD4 + T cells Next, we evaluated if GPI-scFv (X5) would confer the long-term resistance to HIV-1 in human CD4 + T cells.
T98478 19656-19837 Sentence denotes GPI-scFv (X5) confers long-term resistance to HIV-1 in human CD4 + T cells Next, we evaluated if GPI-scFv (X5) would confer the long-term resistance to HIV-1 in human CD4 + T cells.
TextSentencer_T121 19838-19935 Sentence denotes Human CD4 + cell line CEMss was first transfected with a retroviral vector expressing human CCR5.
T28174 19838-19935 Sentence denotes Human CD4 + cell line CEMss was first transfected with a retroviral vector expressing human CCR5.
TextSentencer_T122 19936-20055 Sentence denotes After stable CEMss-CCR5 cells were established, they were further transduced with secretory and GPI-scFv (X5 and AB65).
T37292 19936-20055 Sentence denotes After stable CEMss-CCR5 cells were established, they were further transduced with secretory and GPI-scFv (X5 and AB65).
TextSentencer_T123 20056-20277 Sentence denotes The expression of secretory and GPI-scFvs as well as CD4, CCR5 and CXCR4 in transduced CEMss-CCR5 cells were tested by western blot and immune staining followed by FACS analysis as described above (see Additional File 4).
T74711 20056-20277 Sentence denotes The expression of secretory and GPI-scFvs as well as CD4, CCR5 and CXCR4 in transduced CEMss-CCR5 cells were tested by western blot and immune staining followed by FACS analysis as described above (see Additional File 4).
TextSentencer_T124 20278-20637 Sentence denotes Transduced CEMss-CCR5 cells were then infected with HIV-1 strains Bru-3 and Bru-Yu2 at multiple of infection of 0.01 as described before [23] and cultured in the complete DMEM medium for 75 to 105 days, except for cells transduced with secretory scFvs (AB65 and X5) and infected with Bru-3 (the culture of these cells was terminated on day 27 post infection).
T25376 20278-20637 Sentence denotes Transduced CEMss-CCR5 cells were then infected with HIV-1 strains Bru-3 and Bru-Yu2 at multiple of infection of 0.01 as described before [23] and cultured in the complete DMEM medium for 75 to 105 days, except for cells transduced with secretory scFvs (AB65 and X5) and infected with Bru-3 (the culture of these cells was terminated on day 27 post infection).
TextSentencer_T125 20638-20803 Sentence denotes As shown in Figure 5A and 5B, replication of both HIV-1 Bru-3 and Bru-Yu2 was completely inhibited in cells transduced with GPI-scFv (X5) throughout the experiments.
T98854 20638-20803 Sentence denotes As shown in Figure 5A and 5B, replication of both HIV-1 Bru-3 and Bru-Yu2 was completely inhibited in cells transduced with GPI-scFv (X5) throughout the experiments.
TextSentencer_T126 20804-20952 Sentence denotes In contrast, robust replication of HIV-1 Bru-3 and Bru-Yu2 was observed in cells transduced with secretory scFv (AB65) and GPI-scFv (AB65) controls.
T44912 20804-20952 Sentence denotes In contrast, robust replication of HIV-1 Bru-3 and Bru-Yu2 was observed in cells transduced with secretory scFv (AB65) and GPI-scFv (AB65) controls.
TextSentencer_T127 20953-21116 Sentence denotes For cells transduced with secretory scFv (X5) and infected with HIV-1 Bru-3, HIV-1 replication was as robust as secretory scFv (AB65) and GPI-scFv (AB65) controls.
T77624 20953-21116 Sentence denotes For cells transduced with secretory scFv (X5) and infected with HIV-1 Bru-3, HIV-1 replication was as robust as secretory scFv (AB65) and GPI-scFv (AB65) controls.
TextSentencer_T128 21117-21342 Sentence denotes By contrast, for cells transduced with secretory scFv (X5) and infected with HIV-1 Bru-Yu2, robust HIV-1 replication was observed in the first 6 days and then slowly dropped to the undetectable level on day 51 and thereafter.
T65287 21117-21342 Sentence denotes By contrast, for cells transduced with secretory scFv (X5) and infected with HIV-1 Bru-Yu2, robust HIV-1 replication was observed in the first 6 days and then slowly dropped to the undetectable level on day 51 and thereafter.
TextSentencer_T129 21343-21447 Sentence denotes These data demonstrated that GPI-scFv (X5) completely inhibits the infection of HIV-1 Bru-3 and Bru-Yu2.
T43900 21343-21447 Sentence denotes These data demonstrated that GPI-scFv (X5) completely inhibits the infection of HIV-1 Bru-3 and Bru-Yu2.
TextSentencer_T130 21448-21503 Sentence denotes By so doing it maintains long-term resistance to HIV-1.
T62433 21448-21503 Sentence denotes By so doing it maintains long-term resistance to HIV-1.
TextSentencer_T131 21504-21717 Sentence denotes On the contrary, secretory scFv (X5) cannot inhibit the infection and replication of fast replicating HIV-1 like Bru-3, but can partially inhibits the replication of relatively slow replicating HIV-1 like Bru-Yu2.
T70153 21504-21717 Sentence denotes On the contrary, secretory scFv (X5) cannot inhibit the infection and replication of fast replicating HIV-1 like Bru-3, but can partially inhibits the replication of relatively slow replicating HIV-1 like Bru-Yu2.
TextSentencer_T132 21718-21927 Sentence denotes To evaluate the effect of GPI-scFv (X5) on HIV-1 envelope-mediated cell-cell fusion, the GPI-scFv (X5 and AB65)-transduced CEMss-CCR5 cells were co-cultured with 69TiRevEnv cells as previously described [44] .
T75114 21718-21927 Sentence denotes To evaluate the effect of GPI-scFv (X5) on HIV-1 envelope-mediated cell-cell fusion, the GPI-scFv (X5 and AB65)-transduced CEMss-CCR5 cells were co-cultured with 69TiRevEnv cells as previously described [44] .
TextSentencer_T133 21928-22003 Sentence denotes The latter contains a HIV-1 envelope gene (pLAI3) under a Tet-off promoter.
T12078 21928-22003 Sentence denotes The latter contains a HIV-1 envelope gene (pLAI3) under a Tet-off promoter.
TextSentencer_T134 22004-22369 Sentence denotes In the presence of tetracycline, binding of tetracycline to Tet transactivator (tTA) causes conformational change of tTA, which blocks tTA binding to the Tet-off promoter and prevents HIV-1 envelope protein expression; in the absence of tetracycline, tTA binds to and transactivates the Tet-off promoter resulting in HIV-1 envelope protein expression ( Figure 5C ).
T52560 22004-22369 Sentence denotes In the presence of tetracycline, binding of tetracycline to Tet transactivator (tTA) causes conformational change of tTA, which blocks tTA binding to the Tet-off promoter and prevents HIV-1 envelope protein expression; in the absence of tetracycline, tTA binds to and transactivates the Tet-off promoter resulting in HIV-1 envelope protein expression ( Figure 5C ).
TextSentencer_T135 22370-22528 Sentence denotes Co-culturing GPI-scFv (AB65 and X5)-transduced CEMss-CCR5 cells with tetracycline-treated 69TiRevEnv cells results in no cell-cell fusion ( Figure 5D and 5E).
T2500 22370-22528 Sentence denotes Co-culturing GPI-scFv (AB65 and X5)-transduced CEMss-CCR5 cells with tetracycline-treated 69TiRevEnv cells results in no cell-cell fusion ( Figure 5D and 5E).
TextSentencer_T136 22529-22693 Sentence denotes In contrast, coculturing GPI-scFv (AB65)-transduced CEMss-CCR5 cells with tetracycline-untreated 69TiRevEnv cells results in massive cell-cell fusion ( Figure 5F ).
T10071 22529-22693 Sentence denotes In contrast, coculturing GPI-scFv (AB65)-transduced CEMss-CCR5 cells with tetracycline-untreated 69TiRevEnv cells results in massive cell-cell fusion ( Figure 5F ).
TextSentencer_T137 22694-22748 Sentence denotes The fusion begins after 6 hours and peaks at 20 hours.
T37622 22694-22748 Sentence denotes The fusion begins after 6 hours and peaks at 20 hours.
TextSentencer_T138 22749-22924 Sentence denotes Importantly, no cellcell fusion was observed after 20 hour's co-culturing GPI-scFv (X5)-transduced CEMss-CCR5 cells with tetracycline-untreated 69TiRevEnv cells ( Figure 5G ).
T33136 22749-22924 Sentence denotes Importantly, no cellcell fusion was observed after 20 hour's co-culturing GPI-scFv (X5)-transduced CEMss-CCR5 cells with tetracycline-untreated 69TiRevEnv cells ( Figure 5G ).
TextSentencer_T139 22925-22980 Sentence denotes The experiment was repeated twice with similar results.
T85558 22925-22980 Sentence denotes The experiment was repeated twice with similar results.
TextSentencer_T140 22981-23091 Sentence denotes Thus, these data demonstrated that GPI-scFv (X5) completely inhibits HIV-1 envelope-mediated cell-cell fusion.
T24245 22981-23091 Sentence denotes Thus, these data demonstrated that GPI-scFv (X5) completely inhibits HIV-1 envelope-mediated cell-cell fusion.
TextSentencer_T141 23092-23252 Sentence denotes To test the effect of GPI-scFv (X5) on the infection of HIV-1 captured and transferred by human DCs, monocyte-derived human DCs were incubated with HIV-1 NL4-3.
T1603 23092-23252 Sentence denotes To test the effect of GPI-scFv (X5) on the infection of HIV-1 captured and transferred by human DCs, monocyte-derived human DCs were incubated with HIV-1 NL4-3.
TextSentencer_T142 23253-23311 Sentence denotes Cells were then washed extensively to remove free viruses.
T2319 23253-23311 Sentence denotes Cells were then washed extensively to remove free viruses.
TextSentencer_T143 23312-23410 Sentence denotes Infected DCs were then co-cultured with GPI-scFv (X5 and AB65) transduced CEMss cells for 14 days.
T69202 23312-23410 Sentence denotes Infected DCs were then co-cultured with GPI-scFv (X5 and AB65) transduced CEMss cells for 14 days.
TextSentencer_T144 23411-23480 Sentence denotes HIV-1 replication was measured by HIV-1 p24 assay as described above.
T96600 23411-23480 Sentence denotes HIV-1 replication was measured by HIV-1 p24 assay as described above.
TextSentencer_T145 23481-23671 Sentence denotes As shown Figure 5H , coculturing GPI-scFv (AB65)-transduced CEMss cells with HIV-1 infected monocyte-derived human DCs results in high p24 expression, indicating robust replication of HIV-1.
T78628 23481-23671 Sentence denotes As shown Figure 5H , coculturing GPI-scFv (AB65)-transduced CEMss cells with HIV-1 infected monocyte-derived human DCs results in high p24 expression, indicating robust replication of HIV-1.
TextSentencer_T146 23672-23912 Sentence denotes In contrast, co-culturing GPI-scFv (X5)transduced CEMss cells with HIV-1 infected monocytederived human DCs results in very low level of HIV-1 p24 during the first 4 days and drops off thereafter, indicating inhibition of viral replication.
T62018 23672-23912 Sentence denotes In contrast, co-culturing GPI-scFv (X5)transduced CEMss cells with HIV-1 infected monocytederived human DCs results in very low level of HIV-1 p24 during the first 4 days and drops off thereafter, indicating inhibition of viral replication.
TextSentencer_T147 23913-24170 Sentence denotes This low level of HIV-1 replication detected in the coculture of GPI-scFv (X5)-transduced CEMss cells and HIV-1 infected monocyte-derived human DCs likely reflects slow and covert HIV-1 replication in monocyte-derived human DCs as previously reported [45] .
T8637 23913-24170 Sentence denotes This low level of HIV-1 replication detected in the coculture of GPI-scFv (X5)-transduced CEMss cells and HIV-1 infected monocyte-derived human DCs likely reflects slow and covert HIV-1 replication in monocyte-derived human DCs as previously reported [45] .
TextSentencer_T148 24171-24285 Sentence denotes Thus, the data clearly demonstrated that GPI-scFv (X5) can neutralize HIV-1 captured and transferred by human DCs.
T59208 24171-24285 Sentence denotes Thus, the data clearly demonstrated that GPI-scFv (X5) can neutralize HIV-1 captured and transferred by human DCs.
TextSentencer_T149 24286-24520 Sentence denotes Finally we transduced parental CEMss-CCR5 cells and CEMss-CCR5 [GPI-scFvs (AB65 and X5) secretory scFvs (AB65 and X5)] with VSV-G pseudotyped HIV-1 vector expressing enhanced green fluorescent protein (eGFP) as described before [23] .
T42986 24286-24520 Sentence denotes Finally we transduced parental CEMss-CCR5 cells and CEMss-CCR5 [GPI-scFvs (AB65 and X5) secretory scFvs (AB65 and X5)] with VSV-G pseudotyped HIV-1 vector expressing enhanced green fluorescent protein (eGFP) as described before [23] .
TextSentencer_T150 24521-24739 Sentence denotes Because VSV G envelope interacts with lipid moiety in the lipid bilayer of the plasmic membrane, vectors by pass the requirement of the interaction between HIV-1 envelope and its receptor and coreceptor to enter cells.
T74181 24521-24739 Sentence denotes Because VSV G envelope interacts with lipid moiety in the lipid bilayer of the plasmic membrane, vectors by pass the requirement of the interaction between HIV-1 envelope and its receptor and coreceptor to enter cells.
TextSentencer_T151 24740-24874 Sentence denotes We found that in all three doses tested, transducing parental CEMss-CCR5 cells and CEMss-CCR5 [GPI-scFvs (AB65 and X5) secretory scFvs
T37021 24740-24874 Sentence denotes We found that in all three doses tested, transducing parental CEMss-CCR5 cells and CEMss-CCR5 [GPI-scFvs (AB65 and X5) secretory scFvs
TextSentencer_T152 24876-25062 Sentence denotes In this study we demonstrate that by genetically linking scFvs with GPI-attachment signal scFvs are expressed in the lipid raft of plasma membrane through a GPI anchor (Figure 1 and 2) .
T59335 24876-25062 Sentence denotes In this study we demonstrate that by genetically linking scFvs with GPI-attachment signal scFvs are expressed in the lipid raft of plasma membrane through a GPI anchor (Figure 1 and 2) .
TextSentencer_T153 25063-25271 Sentence denotes GPI-scFvs, but not secretory scFvs, of the antibodies (AB32, TG15, 48d and X5) that recognize transiently-exposed epitopes on HIV-1 envelope spike neutralize HIV-1 with various degrees of breadth and potency.
T89857 25063-25271 Sentence denotes GPI-scFvs, but not secretory scFvs, of the antibodies (AB32, TG15, 48d and X5) that recognize transiently-exposed epitopes on HIV-1 envelope spike neutralize HIV-1 with various degrees of breadth and potency.
TextSentencer_T154 25272-25404 Sentence denotes Among them, GPI-scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 (Figures 3) .
T51785 25272-25404 Sentence denotes Among them, GPI-scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 (Figures 3) .
TextSentencer_T155 25405-25548 Sentence denotes Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart (Figures 3) .
T19340 25405-25548 Sentence denotes Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart (Figures 3) .
TextSentencer_T156 25549-25799 Sentence denotes Importantly, the expression of GPI-scFv (X5) on the surface of human CD4 + T cells confers long-term resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion and the infection of HIV-1 captured and transferred by human DCs (Figure 5 ).
T51064 25549-25799 Sentence denotes Importantly, the expression of GPI-scFv (X5) on the surface of human CD4 + T cells confers long-term resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion and the infection of HIV-1 captured and transferred by human DCs (Figure 5 ).
TextSentencer_T157 25800-26029 Sentence denotes Thus, targeting scFv of antibody molecules in the lipid rafts of plasma membranes of HIV-1 susceptible cells through a GPI anchor is an effective way to capture transiently exposed neutralization epitopes of HIV-1 envelope spike.
T38919 25800-26029 Sentence denotes Thus, targeting scFv of antibody molecules in the lipid rafts of plasma membranes of HIV-1 susceptible cells through a GPI anchor is an effective way to capture transiently exposed neutralization epitopes of HIV-1 envelope spike.
TextSentencer_T158 26030-26183 Sentence denotes GPI-scFv (X5) with such remarkable breadth and potency should have a potential to be developed into an anti-viral agent for HIV-1 prevention and therapy.
T38283 26030-26183 Sentence denotes GPI-scFv (X5) with such remarkable breadth and potency should have a potential to be developed into an anti-viral agent for HIV-1 prevention and therapy.
TextSentencer_T159 26184-26441 Sentence denotes For example, similar to those recently reported by DiGiusto et al. [46] , GPI-scFv (X5) could be delivered into hematopoietic progenitor cells of HIV-1 patients ex vivo through lentiviral vector and transduced cells could then be transfused to the patients.
T32113 26184-26441 Sentence denotes For example, similar to those recently reported by DiGiusto et al. [46] , GPI-scFv (X5) could be delivered into hematopoietic progenitor cells of HIV-1 patients ex vivo through lentiviral vector and transduced cells could then be transfused to the patients.
TextSentencer_T160 26442-26829 Sentence denotes However, in order to achieve clinical efficacy with this gene therapy approach, many hurdles, such as low degree of transduction efficiency and engraftment, difficulty in maintenance of self renewal as well as hematopoietic linage cell differentiation of transduced hematopoietic progenitor cells, unsustainable transgene expression, potential insertion mutagenesis, have to be overcome.
T57410 26442-26829 Sentence denotes However, in order to achieve clinical efficacy with this gene therapy approach, many hurdles, such as low degree of transduction efficiency and engraftment, difficulty in maintenance of self renewal as well as hematopoietic linage cell differentiation of transduced hematopoietic progenitor cells, unsustainable transgene expression, potential insertion mutagenesis, have to be overcome.
TextSentencer_T161 26830-27111 Sentence denotes Interestingly, although their level of expression on the cell surface is similar ( Figure 1C ), 6 anti-HIV-1 gp120 or gp41 GPI-scFvs tested in this study display the wide spectrum of breadth and potency against 11 HIV-1 pseudotypes and 6 wild type HIV-1 strains tested (Figure 3) .
T47427 26830-27111 Sentence denotes Interestingly, although their level of expression on the cell surface is similar ( Figure 1C ), 6 anti-HIV-1 gp120 or gp41 GPI-scFvs tested in this study display the wide spectrum of breadth and potency against 11 HIV-1 pseudotypes and 6 wild type HIV-1 strains tested (Figure 3) .
TextSentencer_T162 27112-27360 Sentence denotes At one end of the spectrum, GPI-scFv (AB31) does not show significant neutralization activity against any of these HIV-1 viruses; while on the other end of the spectrum, GPI-scFv (X5) neutralizes all HIV-1 viruses with remarkable degree of potency.
T6215 27112-27360 Sentence denotes At one end of the spectrum, GPI-scFv (AB31) does not show significant neutralization activity against any of these HIV-1 viruses; while on the other end of the spectrum, GPI-scFv (X5) neutralizes all HIV-1 viruses with remarkable degree of potency.
TextSentencer_T163 27361-27477 Sentence denotes Between these two extremes, GPI-scFvs (AB32, TG15, 4E10 and 48d) show a progressive increase of breadth and potency.
T89370 27361-27477 Sentence denotes Between these two extremes, GPI-scFvs (AB32, TG15, 4E10 and 48d) show a progressive increase of breadth and potency.
TextSentencer_T164 27478-27703 Sentence denotes Furthermore, although they both recognize CD4-induced epitopes, GPI-scFv (48d) and GPI-scFv (X5) show different potency against HIV-1 strains from clade C, but similar potency against HIV-1 strains from clades A, B, B' and E.
T89897 27478-27703 Sentence denotes Furthermore, although they both recognize CD4-induced epitopes, GPI-scFv (48d) and GPI-scFv (X5) show different potency against HIV-1 strains from clade C, but similar potency against HIV-1 strains from clades A, B, B' and E.
TextSentencer_T165 27704-27938 Sentence denotes Thus these results strongly suggest that the breadth and potency of a given GPI-scFv does not just depend on local concentration, but it is dependent on the fine specificity of the transientlyexposed neutralization epitope it targets.
T50790 27704-27938 Sentence denotes Thus these results strongly suggest that the breadth and potency of a given GPI-scFv does not just depend on local concentration, but it is dependent on the fine specificity of the transientlyexposed neutralization epitope it targets.
TextSentencer_T166 27939-28067 Sentence denotes In this study, we also show that potent inhibition of HIV-1 by GPI-scFv (X5) does not depend on additional sCD4 (see Figure 4 ).
T46608 27939-28067 Sentence denotes In this study, we also show that potent inhibition of HIV-1 by GPI-scFv (X5) does not depend on additional sCD4 (see Figure 4 ).
TextSentencer_T167 28068-28136 Sentence denotes We believe that this likely has something to do with the X5 epitope.
T15199 28068-28136 Sentence denotes We believe that this likely has something to do with the X5 epitope.
TextSentencer_T168 28137-28388 Sentence denotes The X5 epitope resides within a conserved region (amino acid residues 417 to 434) of the gp120 core, particularly in the vicinity of amino acid residues at positions 423 and 432, which is in the proximity of the CD4 and coreceptor binding sites [35] .
T20219 28137-28388 Sentence denotes The X5 epitope resides within a conserved region (amino acid residues 417 to 434) of the gp120 core, particularly in the vicinity of amino acid residues at positions 423 and 432, which is in the proximity of the CD4 and coreceptor binding sites [35] .
TextSentencer_T169 28389-28668 Sentence denotes According to the crystal structure of gp120 core with or without sCD4 [41, 42] as well as crystal structure of a complex containing V3-containing gp120 core, the CD4 receptor and the Fab (X5) [47] , the amino acid residues 417 to 434 reside in β19, β20 and β21 of the gp120 core.
T22909 28389-28668 Sentence denotes According to the crystal structure of gp120 core with or without sCD4 [41, 42] as well as crystal structure of a complex containing V3-containing gp120 core, the CD4 receptor and the Fab (X5) [47] , the amino acid residues 417 to 434 reside in β19, β20 and β21 of the gp120 core.
TextSentencer_T170 28669-28719 Sentence denotes The β20 and the β21 form an anti-parallel β sheet.
T76835 28669-28719 Sentence denotes The β20 and the β21 form an anti-parallel β sheet.
TextSentencer_T171 28720-28865 Sentence denotes Without adding sCD4, β19 and β20/β21 are away from each other, the β19 is covered by V3 loop and the β20/β21 is partially covered by V1/ V2 loop.
T58615 28720-28865 Sentence denotes Without adding sCD4, β19 and β20/β21 are away from each other, the β19 is covered by V3 loop and the β20/β21 is partially covered by V1/ V2 loop.
TextSentencer_T172 28866-28941 Sentence denotes Because of this, secretory scFv (X5) does not have neutralization activity.
T95695 28866-28941 Sentence denotes Because of this, secretory scFv (X5) does not have neutralization activity.
TextSentencer_T173 28942-29011 Sentence denotes In contrast, adding sCD4 induces a CD4-induced conformation of gp120.
T56143 28942-29011 Sentence denotes In contrast, adding sCD4 induces a CD4-induced conformation of gp120.
TextSentencer_T174 29012-29130 Sentence denotes In this conformation, the β19 and the β20/β21 are not only totally exposed, but also become close to each other [42] .
T52214 29012-29130 Sentence denotes In this conformation, the β19 and the β20/β21 are not only totally exposed, but also become close to each other [42] .
TextSentencer_T175 29131-29232 Sentence denotes As a result, adding sCD4 greatly enhances neutralization activity of secretory scFv (X5) (Figure 4 ).
T94139 29131-29232 Sentence denotes As a result, adding sCD4 greatly enhances neutralization activity of secretory scFv (X5) (Figure 4 ).
TextSentencer_T176 29233-29452 Sentence denotes However, it is very plausible that even in the presence of sCD4 CD4-induced conformation of the HIV-1 spike is transient, so that secretory scFv (X5) may not be able to catch all X5 epitopes on the spikes of the virion.
T1252 29233-29452 Sentence denotes However, it is very plausible that even in the presence of sCD4 CD4-induced conformation of the HIV-1 spike is transient, so that secretory scFv (X5) may not be able to catch all X5 epitopes on the spikes of the virion.
TextSentencer_T177 29453-29505 Sentence denotes As a result, neutralization activity is not optimal.
T49900 29453-29505 Sentence denotes As a result, neutralization activity is not optimal.
TextSentencer_T178 29506-29729 Sentence denotes GPI-scFv (X5), on the other hand, due to its lipid raft location and local concentration, may have much better access to its transiently-exposed neutralization epitope upon interaction of CD4 with the native envelope spike.
T2640 29506-29729 Sentence denotes GPI-scFv (X5), on the other hand, due to its lipid raft location and local concentration, may have much better access to its transiently-exposed neutralization epitope upon interaction of CD4 with the native envelope spike.
TextSentencer_T179 29730-29877 Sentence denotes Here CD4, a raft-associated protein, resides in the lipid raft of the plasma membrane of target cells, which is the same location as GPI-scFv (X5).
T66754 29730-29877 Sentence denotes Here CD4, a raft-associated protein, resides in the lipid raft of the plasma membrane of target cells, which is the same location as GPI-scFv (X5).
TextSentencer_T180 29878-30054 Sentence denotes As a result, GPI-scFv (X5) may quickly and efficiently capture the CD4-induced neutralization epitope and completely block the envelope spike in a pre-fusion intermediate step.
T61876 29878-30054 Sentence denotes As a result, GPI-scFv (X5) may quickly and efficiently capture the CD4-induced neutralization epitope and completely block the envelope spike in a pre-fusion intermediate step.
TextSentencer_T181 30055-30148 Sentence denotes However, the underlying mechanism of great potency of GPI-scFv (X5) remains to be elucidated.
T58853 30055-30148 Sentence denotes However, the underlying mechanism of great potency of GPI-scFv (X5) remains to be elucidated.
TextSentencer_T182 30149-30215 Sentence denotes There are at least two implications of the findings in this study.
T15699 30149-30215 Sentence denotes There are at least two implications of the findings in this study.
TextSentencer_T183 30216-30432 Sentence denotes First, given the promising results obtained in this study, it is plausible to develop GPI-scFvs into a standard approach for identifying antibodies that recognize transiently-exposed epitopes on HIV-1 envelope spike.
T2175 30216-30432 Sentence denotes First, given the promising results obtained in this study, it is plausible to develop GPI-scFvs into a standard approach for identifying antibodies that recognize transiently-exposed epitopes on HIV-1 envelope spike.
TextSentencer_T184 30433-30689 Sentence denotes It is conceivable that for many anti-HIV-1 gp120 and gp41 antibodies that have no or poor neutralization activity against native envelope spikes, GPI-scFvs of these antibodies can be made and tested against a panel of different HIV-1 variants and subtypes.
T75878 30433-30689 Sentence denotes It is conceivable that for many anti-HIV-1 gp120 and gp41 antibodies that have no or poor neutralization activity against native envelope spikes, GPI-scFvs of these antibodies can be made and tested against a panel of different HIV-1 variants and subtypes.
TextSentencer_T185 30690-30951 Sentence denotes If some of these GPI-scFvs exhibit good neutralization activity, one can then use these GPI-scFvs in combination with epitope mapping methods (such as alanine-scanning mutants) to identify transiently-exposed neutralization epitopes in the HIV-1 envelope spike.
T18049 30690-30951 Sentence denotes If some of these GPI-scFvs exhibit good neutralization activity, one can then use these GPI-scFvs in combination with epitope mapping methods (such as alanine-scanning mutants) to identify transiently-exposed neutralization epitopes in the HIV-1 envelope spike.
TextSentencer_T186 30952-31344 Sentence denotes Second, it is also plausible that GPI-scFvs be used to identify antibodies that recognize transiently-exposed epitopes and then to identify transiently-exposed neutralization epitopes of other viruses, particularly of those enveloped viruses whose entry into target cells depend on lipid raft and the interaction of those envelope glycoprotein with receptor results in conformational changes.
T86784 30952-31344 Sentence denotes Second, it is also plausible that GPI-scFvs be used to identify antibodies that recognize transiently-exposed epitopes and then to identify transiently-exposed neutralization epitopes of other viruses, particularly of those enveloped viruses whose entry into target cells depend on lipid raft and the interaction of those envelope glycoprotein with receptor results in conformational changes.
TextSentencer_T187 31345-31535 Sentence denotes It has been shown that glycoproteins of influenza virus, murine leukemia virus, measles virus and Ebola virus are associated with the lipid raft of plasma membrane [48] [49] [50] [51] [52] .
T23530 31345-31535 Sentence denotes It has been shown that glycoproteins of influenza virus, murine leukemia virus, measles virus and Ebola virus are associated with the lipid raft of plasma membrane [48] [49] [50] [51] [52] .
TextSentencer_T188 31536-31794 Sentence denotes The entry of many enveloped viruses, such as vaccinia virus [53] , severe acute respiratory syndrome-coronavirus [54] , duck hepatitis B virus [55] , murine leukemia virus [56] and herpes simplex virus [57] , requires cholesterol in the target cell membrane.
T23702 31536-31794 Sentence denotes The entry of many enveloped viruses, such as vaccinia virus [53] , severe acute respiratory syndrome-coronavirus [54] , duck hepatitis B virus [55] , murine leukemia virus [56] and herpes simplex virus [57] , requires cholesterol in the target cell membrane.
TextSentencer_T189 31795-32010 Sentence denotes Therefore, development of GPI-scFvs that are derived from antibodies that recognize transiently-exposed epitopes in glycoproteins of these viruses may also be an effective way to inhibit cell entry of these viruses.
T44337 31795-32010 Sentence denotes Therefore, development of GPI-scFvs that are derived from antibodies that recognize transiently-exposed epitopes in glycoproteins of these viruses may also be an effective way to inhibit cell entry of these viruses.
TextSentencer_T190 32011-32154 Sentence denotes GPI anchored scFvs that recognize different transientlyexposed epitopes on HIV-1 envelope spike exhibit various degrees of breadth and potency.
T50064 32011-32154 Sentence denotes GPI anchored scFvs that recognize different transientlyexposed epitopes on HIV-1 envelope spike exhibit various degrees of breadth and potency.
TextSentencer_T191 32155-32347 Sentence denotes Among them, GPI-scFv (X5) that recognizes the epitope near the CD4 binding site and coreceptor (CCR5) binding site, exhibited greatest potency and breadth against multiclades of HIV-1 strains.
T23658 32155-32347 Sentence denotes Among them, GPI-scFv (X5) that recognizes the epitope near the CD4 binding site and coreceptor (CCR5) binding site, exhibited greatest potency and breadth against multiclades of HIV-1 strains.
TextSentencer_T192 32348-32553 Sentence denotes Our data therefore demonstrate that GPI-scFvs may be developed as a useful tool to identify antibodies that recognize transiently-exposed epitopes in glycoproteins spike of HIV and other enveloped viruses.
T52741 32348-32553 Sentence denotes Our data therefore demonstrate that GPI-scFvs may be developed as a useful tool to identify antibodies that recognize transiently-exposed epitopes in glycoproteins spike of HIV and other enveloped viruses.
TextSentencer_T193 32554-32676 Sentence denotes GPI-scFvs derived from such antibodies could be an effective way to inhibit cell entry of HIV and other enveloped viruses.
T26118 32554-32676 Sentence denotes GPI-scFvs derived from such antibodies could be an effective way to inhibit cell entry of HIV and other enveloped viruses.
TextSentencer_T194 32677-32830 Sentence denotes Proviral plasmids pBru-3, pBru-Yu2, pAD8, pNL4-3 and pMj4 were obtained from the NIH AIDS Research and Reference Reagent Program (ARRRP, Germantown, MD).
T46510 32677-32830 Sentence denotes Proviral plasmids pBru-3, pBru-Yu2, pAD8, pNL4-3 and pMj4 were obtained from the NIH AIDS Research and Reference Reagent Program (ARRRP, Germantown, MD).
TextSentencer_T195 32831-33129 Sentence denotes Infectious virus stocks of HIV-1 Bru-3 and NL4-3 (X4 tropic, subtype B), Bru-Yu2, and AD8 (R5 tropic, subtype B) and Mj4 (R5 tropic, subtype C) were produced by transfecting into 293T cells using a calcium phosphate precipitation or a Fugene-6 reagent method (Roche Diagnostics, Mannheim, Germany).
T60156 32831-33129 Sentence denotes Infectious virus stocks of HIV-1 Bru-3 and NL4-3 (X4 tropic, subtype B), Bru-Yu2, and AD8 (R5 tropic, subtype B) and Mj4 (R5 tropic, subtype C) were produced by transfecting into 293T cells using a calcium phosphate precipitation or a Fugene-6 reagent method (Roche Diagnostics, Mannheim, Germany).
TextSentencer_T196 33130-33317 Sentence denotes Virus stocks were then propagated by infecting phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMCs) with these infectious clones as described before [24] .
T65387 33130-33317 Sentence denotes Virus stocks were then propagated by infecting phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMCs) with these infectious clones as described before [24] .
TextSentencer_T197 33318-33516 Sentence denotes Primary HIV-1 isolates RJ407 and JS-JCD were generated by infecting PHA-stimulated human PBMCs with plasmas of two subjects in a cohort of individuals who was recruited for a study of HIV-1 fitness.
T37101 33318-33516 Sentence denotes Primary HIV-1 isolates RJ407 and JS-JCD were generated by infecting PHA-stimulated human PBMCs with plasmas of two subjects in a cohort of individuals who was recruited for a study of HIV-1 fitness.
TextSentencer_T198 33517-33657 Sentence denotes Informed consent was obtained from participants in accordance with the Institutional Review Boards of the Huashan Hospital, Shanghai, China.
T65147 33517-33657 Sentence denotes Informed consent was obtained from participants in accordance with the Institutional Review Boards of the Huashan Hospital, Shanghai, China.
TextSentencer_T199 33658-33778 Sentence denotes All virus stocks (Bru-3, NL4-3, Bru-Yu2, AD8, Mj4, RJ407, and JS-JCD) were stored in aliquots at a -80°C degree freezer.
T84817 33658-33778 Sentence denotes All virus stocks (Bru-3, NL4-3, Bru-Yu2, AD8, Mj4, RJ407, and JS-JCD) were stored in aliquots at a -80°C degree freezer.
TextSentencer_T200 33779-33871 Sentence denotes The titers of the stocks were determined by end-point titration on TZM.bl cells (see below).
T6284 33779-33871 Sentence denotes The titers of the stocks were determined by end-point titration on TZM.bl cells (see below).
TextSentencer_T201 33872-34197 Sentence denotes The packaging cell line 293T was purchased from Invitrogen Life Technologies (San Diego, CA) and maintained in complete DMEM medium [i.e. high glucose DMEM supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin (100 U/ml), streptomycin (100 μg/ml)] plus G418 (500 μg/ml) (Invitrogen Life Technologies).
T51355 33872-34197 Sentence denotes The packaging cell line 293T was purchased from Invitrogen Life Technologies (San Diego, CA) and maintained in complete DMEM medium [i.e. high glucose DMEM supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin (100 U/ml), streptomycin (100 μg/ml)] plus G418 (500 μg/ml) (Invitrogen Life Technologies).
TextSentencer_T202 34198-34372 Sentence denotes Human CD4 + T cell line CEMss was obtained from Dr. Jon Allan at the Department of Virology and Immunology, the Southwest Foundation for Biomedical Research, San Antonio, TX.
T86434 34198-34372 Sentence denotes Human CD4 + T cell line CEMss was obtained from Dr. Jon Allan at the Department of Virology and Immunology, the Southwest Foundation for Biomedical Research, San Antonio, TX.
TextSentencer_T203 34373-34452 Sentence denotes TZM.bl and 69Tirev/env cells were obtained from NIH ARRRP, as contributed by J.
T65351 34373-34452 Sentence denotes TZM.bl and 69Tirev/env cells were obtained from NIH ARRRP, as contributed by J.
TextSentencer_T204 34453-34466 Sentence denotes Kappes and X.
T41490 34453-34466 Sentence denotes Kappes and X.
TextSentencer_T205 34467-34485 Sentence denotes Wu [58, 59] and H.
T37921 34467-34485 Sentence denotes Wu [58, 59] and H.
TextSentencer_T206 34486-34509 Sentence denotes Yu [44] , respectively.
T87342 34486-34509 Sentence denotes Yu [44] , respectively.
TextSentencer_T207 34510-34572 Sentence denotes CEMss cells and TZM.bl cells were maintained in complete DMEM.
T98120 34510-34572 Sentence denotes CEMss cells and TZM.bl cells were maintained in complete DMEM.
TextSentencer_T208 34573-34644 Sentence denotes 69TiRev/Env cells were maintained in complete DMEM plus 0.2 mg/ml G418.
T65400 34573-34644 Sentence denotes 69TiRev/Env cells were maintained in complete DMEM plus 0.2 mg/ml G418.
TextSentencer_T209 34645-35016 Sentence denotes Drosophila Schneider 2 (S2) cells were purchased from Invitrogen Life Technologies and maintained in 75 cm flask (Corning) at the cell density between 0.2 to 2 × 10 6 cells per ml in the Express FIVE® SFM medium (Invitrogen Life Technologies) supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin and 100 mg/L L-glutamine at 28°C without additional CO 2 .
T68130 34645-35016 Sentence denotes Drosophila Schneider 2 (S2) cells were purchased from Invitrogen Life Technologies and maintained in 75 cm flask (Corning) at the cell density between 0.2 to 2 × 10 6 cells per ml in the Express FIVE® SFM medium (Invitrogen Life Technologies) supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin and 100 mg/L L-glutamine at 28°C without additional CO 2 .
TextSentencer_T210 35017-35353 Sentence denotes To construct the scFv/IgG3 hinge/his-tag/DAF, a 143-bp DNA fragment containing a 8-bp sequence of 3'end of human IgG3 hinge region, a 123-bp sequence encoding a 6 x his tag, the C-terminal 34 amino acid residues of DAF [37] and a stop codon, and a 12-bp sequence of a Sal I site and additional 6 bp were linked by a recursive PCR [60] .
T40654 35017-35353 Sentence denotes To construct the scFv/IgG3 hinge/his-tag/DAF, a 143-bp DNA fragment containing a 8-bp sequence of 3'end of human IgG3 hinge region, a 123-bp sequence encoding a 6 x his tag, the C-terminal 34 amino acid residues of DAF [37] and a stop codon, and a 12-bp sequence of a Sal I site and additional 6 bp were linked by a recursive PCR [60] .
TextSentencer_T211 35354-35687 Sentence denotes Another DNA fragment containing a 12-bp sequence of a Bgl II site and additional 6 bp, a sequence encoding the signal peptide, anti-HIV-1 gp41 scFv (TG15) and 11 residues of human IgG3 hinge region, and an 8-bp sequence of the 5'end of the his tag was amplified by PCR using previously generated pLNCX-TCR-m-scFv as a template [23] .
T533 35354-35687 Sentence denotes Another DNA fragment containing a 12-bp sequence of a Bgl II site and additional 6 bp, a sequence encoding the signal peptide, anti-HIV-1 gp41 scFv (TG15) and 11 residues of human IgG3 hinge region, and an 8-bp sequence of the 5'end of the his tag was amplified by PCR using previously generated pLNCX-TCR-m-scFv as a template [23] .
TextSentencer_T212 35688-35846 Sentence denotes These two fragments were designed in such a way that there were 16 nucleotides overlapping between the 3'end of scFv/hinge and 5'end of his tag/DAF fragments.
T46224 35688-35846 Sentence denotes These two fragments were designed in such a way that there were 16 nucleotides overlapping between the 3'end of scFv/hinge and 5'end of his tag/DAF fragments.
TextSentencer_T213 35847-36083 Sentence denotes In addition, an Xba I site was inserted between the sequences of the signal peptide and the scFv and a Xma I site was inserted between the sequences of scFv and IgG3 hinge region to facilitate cloning process of other scFvs (see below).
T20342 35847-36083 Sentence denotes In addition, an Xba I site was inserted between the sequences of the signal peptide and the scFv and a Xma I site was inserted between the sequences of scFv and IgG3 hinge region to facilitate cloning process of other scFvs (see below).
TextSentencer_T214 36084-36194 Sentence denotes The whole fragment encoding the entire scFv(TG15)/hinge/his-tag/ DAF was then generated by an overlapping PCR.
T13899 36084-36194 Sentence denotes The whole fragment encoding the entire scFv(TG15)/hinge/his-tag/ DAF was then generated by an overlapping PCR.
TextSentencer_T215 36195-36469 Sentence denotes The amplified cDNA was then ligated in a TA vector system (Invitrogen Life Technologies) for sequence analysis. cDNA containing the correct scFv(TG15)/hinge/his-tag/ DAF was cloned into the Bam HI and Sal I sites of lentiviral transfer vector pRRLsin-18.PPT.hPGK.Wpre [61] .
T89952 36195-36469 Sentence denotes The amplified cDNA was then ligated in a TA vector system (Invitrogen Life Technologies) for sequence analysis. cDNA containing the correct scFv(TG15)/hinge/his-tag/ DAF was cloned into the Bam HI and Sal I sites of lentiviral transfer vector pRRLsin-18.PPT.hPGK.Wpre [61] .
TextSentencer_T216 36470-36580 Sentence denotes The resulting lentiviral transfer construct was designated as pRRL-scFv(TG15)/hinge/his-tag/DAF ( Figure 1A) .
T74256 36470-36580 Sentence denotes The resulting lentiviral transfer construct was designated as pRRL-scFv(TG15)/hinge/his-tag/DAF ( Figure 1A) .
TextSentencer_T217 36581-36753 Sentence denotes To construct genes encoding scFvs (AB31, AB32, 4E10, X5 and 48d), overlapping primers for the recursive PCR were synthesized according to the published sequences [34, 62] .
T48380 36581-36753 Sentence denotes To construct genes encoding scFvs (AB31, AB32, 4E10, X5 and 48d), overlapping primers for the recursive PCR were synthesized according to the published sequences [34, 62] .
TextSentencer_T218 36754-36916 Sentence denotes The end primers of recursive PCR were designed in such a way that the Xba I and the Xma I sites were introduced at 5' and 3' ends of the scFv genes, respectively.
T589 36754-36916 Sentence denotes The end primers of recursive PCR were designed in such a way that the Xba I and the Xma I sites were introduced at 5' and 3' ends of the scFv genes, respectively.
TextSentencer_T219 36917-37119 Sentence denotes The gene encoding scFv (AB65) was derived from the VH and VL fragments of a human monoclonal antibody generated from convalescent plasma of H5N1 avian influenza virus infected patient in our laboratory.
T86284 36917-37119 Sentence denotes The gene encoding scFv (AB65) was derived from the VH and VL fragments of a human monoclonal antibody generated from convalescent plasma of H5N1 avian influenza virus infected patient in our laboratory.
TextSentencer_T220 37120-37328 Sentence denotes The amplified genes encoding scFvs were ligated in a TA vector system for sequencing. cDNA containing the correct scFvs were then inserted into the Xba I and Xma I sites of pRRL-scFv (TG15)/hinge/his-tag/DAF.
T1063 37120-37328 Sentence denotes The amplified genes encoding scFvs were ligated in a TA vector system for sequencing. cDNA containing the correct scFvs were then inserted into the Xba I and Xma I sites of pRRL-scFv (TG15)/hinge/his-tag/DAF.
TextSentencer_T221 37329-37509 Sentence denotes The resulting lentiviral transfer constructs were designated as pRRL-scFv(AB31, AB32, 4E10, X5, 48d and AB65)/hinge/his-tag/DAF, respectively (see Figure 1A and Additional File 1).
T99423 37329-37509 Sentence denotes The resulting lentiviral transfer constructs were designated as pRRL-scFv(AB31, AB32, 4E10, X5, 48d and AB65)/hinge/his-tag/DAF, respectively (see Figure 1A and Additional File 1).
TextSentencer_T222 37510-37744 Sentence denotes To construct a secretory form of the scFvs, a gene encoding sequence containing the signal peptide, scFv (TG15), IgG3 hinge, his-tag and a stop codon was first amplified by PCR using the pRRL-scFv(TG15)/hinge/histag/DAF as a template.
T62122 37510-37744 Sentence denotes To construct a secretory form of the scFvs, a gene encoding sequence containing the signal peptide, scFv (TG15), IgG3 hinge, his-tag and a stop codon was first amplified by PCR using the pRRL-scFv(TG15)/hinge/histag/DAF as a template.
TextSentencer_T223 37745-37929 Sentence denotes The correct sequence was inserted into the Bam HI and the Sal I sites of pRRL-scFv(TG15)/ hinge/his-tag/DAF to generate the lentiviral transfer construct pRRL-scFv(TG15)/hinge/his-tag.
T6401 37745-37929 Sentence denotes The correct sequence was inserted into the Bam HI and the Sal I sites of pRRL-scFv(TG15)/ hinge/his-tag/DAF to generate the lentiviral transfer construct pRRL-scFv(TG15)/hinge/his-tag.
TextSentencer_T224 37930-38097 Sentence denotes Then sequences encoding each of the other scFvs (AB31, AB32, X5, 4E10, 48d and AB65) were inserted into the Xba I and the Xma I sites of pRRL-scFv(TG15)/hinge/his-tag.
T73245 37930-38097 Sentence denotes Then sequences encoding each of the other scFvs (AB31, AB32, X5, 4E10, 48d and AB65) were inserted into the Xba I and the Xma I sites of pRRL-scFv(TG15)/hinge/his-tag.
TextSentencer_T225 38098-38272 Sentence denotes The resulting lentiviral transfer constructs were designated as pRRL-scFv(AB31, AB32, X5, 4E10, 48d and AB65)/hinge/ his-tag, respectively ( Figure 1A and Additional File 1).
T55529 38098-38272 Sentence denotes The resulting lentiviral transfer constructs were designated as pRRL-scFv(AB31, AB32, X5, 4E10, 48d and AB65)/hinge/ his-tag, respectively ( Figure 1A and Additional File 1).
TextSentencer_T226 38273-38584 Sentence denotes To generate genes encoding soluble human CD4 protein to be produced by Drosophila S2 cells, a gene encoding extracellular domain of the human CD4 protein with a his-tag at its C-terminus was PCR-amplified using total RNA isolated from CEMss cells as a template and ligated into the TA vector as described above.
T78137 38273-38584 Sentence denotes To generate genes encoding soluble human CD4 protein to be produced by Drosophila S2 cells, a gene encoding extracellular domain of the human CD4 protein with a his-tag at its C-terminus was PCR-amplified using total RNA isolated from CEMss cells as a template and ligated into the TA vector as described above.
TextSentencer_T227 38585-38722 Sentence denotes The correct gene sequence was then inserted into Bgl II and BstB I sites of an inducible S2 expression vector pMTbip/V5-His (Invitrogen).
T4787 38585-38722 Sentence denotes The correct gene sequence was then inserted into Bgl II and BstB I sites of an inducible S2 expression vector pMTbip/V5-His (Invitrogen).
TextSentencer_T228 38723-38776 Sentence denotes The resulting plasmid was designated as pMT-bip-sCD4.
T23024 38723-38776 Sentence denotes The resulting plasmid was designated as pMT-bip-sCD4.
TextSentencer_T229 38777-38927 Sentence denotes The gene encoding retroviral envelope 10A1 was PCRamplified from the packaging cell line PT67 originally developed in Dusty Miller's laboratory [39] .
T47444 38777-38927 Sentence denotes The gene encoding retroviral envelope 10A1 was PCRamplified from the packaging cell line PT67 originally developed in Dusty Miller's laboratory [39] .
TextSentencer_T230 38928-39027 Sentence denotes The gene encoding the HIV-1 envelope Yu2 was PCR-amplified from proviral DNA (Bru-Yu2) (see above).
T27703 38928-39027 Sentence denotes The gene encoding the HIV-1 envelope Yu2 was PCR-amplified from proviral DNA (Bru-Yu2) (see above).
TextSentencer_T231 39028-39151 Sentence denotes The correct sequences (10A1 and Yu2) were then cloned into the Sal I and the Bam HI sites of CMV/R expression vector [38] .
T15479 39028-39151 Sentence denotes The correct sequences (10A1 and Yu2) were then cloned into the Sal I and the Bam HI sites of CMV/R expression vector [38] .
TextSentencer_T232 39152-39246 Sentence denotes The resulting plasmids were designated as CMV/ R-env (10A1) and CMV/R-env (Yu2), respectively.
T16516 39152-39246 Sentence denotes The resulting plasmids were designated as CMV/ R-env (10A1) and CMV/R-env (Yu2), respectively.
TextSentencer_T233 39247-39400 Sentence denotes The gene encoding human CCR5 was PCR amplified using total RNA isolated from human PBMCs as a template and ligated into the TA vector as described above.
T11151 39247-39400 Sentence denotes The gene encoding human CCR5 was PCR amplified using total RNA isolated from human PBMCs as a template and ligated into the TA vector as described above.
TextSentencer_T234 39401-39530 Sentence denotes The correct gene sequence was then inserted into Bam HI and Sal I sites of a lentiviral transfer vector pRRLsin-18.PPT.hPGK.Wpre.
T78651 39401-39530 Sentence denotes The correct gene sequence was then inserted into Bam HI and Sal I sites of a lentiviral transfer vector pRRLsin-18.PPT.hPGK.Wpre.
TextSentencer_T235 39531-39603 Sentence denotes The resulting lentiviral transfer construct was designated as pRRL-CCR5.
T685 39531-39603 Sentence denotes The resulting lentiviral transfer construct was designated as pRRL-CCR5.
TextSentencer_T236 39604-39676 Sentence denotes Recombinant lentiviral vectors were generated as described before [23] .
T71223 39604-39676 Sentence denotes Recombinant lentiviral vectors were generated as described before [23] .
TextSentencer_T237 39677-39757 Sentence denotes Briefly, 4 × 10 6 293T cells were seeded onto P-100 dish in 10 ml complete DMEM.
T38492 39677-39757 Sentence denotes Briefly, 4 × 10 6 293T cells were seeded onto P-100 dish in 10 ml complete DMEM.
TextSentencer_T238 39758-40131 Sentence denotes After overnight culture, cells were co-transfected with 20 μg transfer construct (one of pRRL-scFv/hinge/histag/DAFs or pRRL-scFv/hinge/his-tags as well as pRRL-CCR5), 10 μg packaging construct encoding HIV-1 gag/ pol (pLP1), 7.5 μg plasmids encoding VSV-G envelope (pLP/VSVG) and 7.5 μg HIV-1 rev protein (pLP2) (Invitrogen) using a calcium phosphate precipitation method.
T46441 39758-40131 Sentence denotes After overnight culture, cells were co-transfected with 20 μg transfer construct (one of pRRL-scFv/hinge/histag/DAFs or pRRL-scFv/hinge/his-tags as well as pRRL-CCR5), 10 μg packaging construct encoding HIV-1 gag/ pol (pLP1), 7.5 μg plasmids encoding VSV-G envelope (pLP/VSVG) and 7.5 μg HIV-1 rev protein (pLP2) (Invitrogen) using a calcium phosphate precipitation method.
TextSentencer_T239 40132-40259 Sentence denotes Sixteen hours later, culture supernatants were removed and replaced with fresh complete DMEM plus 1 mM sodium butyrate (Sigma).
T26268 40132-40259 Sentence denotes Sixteen hours later, culture supernatants were removed and replaced with fresh complete DMEM plus 1 mM sodium butyrate (Sigma).
TextSentencer_T240 40260-40352 Sentence denotes Eight hours later, supernatants were again removed and replaced with fresh DMEM plus 4% FBS.
T75899 40260-40352 Sentence denotes Eight hours later, supernatants were again removed and replaced with fresh DMEM plus 4% FBS.
TextSentencer_T241 40353-40484 Sentence denotes After another 20 hours, the culture supernatants were harvested and concentrated by ultra-centrifugation as described before [38] .
T19299 40353-40484 Sentence denotes After another 20 hours, the culture supernatants were harvested and concentrated by ultra-centrifugation as described before [38] .
TextSentencer_T242 40485-40594 Sentence denotes The vector pellets were resuspended in a small volume of DMEM and stored in aliquots at -80°C degree freezer.
T46568 40485-40594 Sentence denotes The vector pellets were resuspended in a small volume of DMEM and stored in aliquots at -80°C degree freezer.
TextSentencer_T243 40595-40658 Sentence denotes Vector titers were determined as we previously described [23] .
T98636 40595-40658 Sentence denotes Vector titers were determined as we previously described [23] .
TextSentencer_T244 40659-40753 Sentence denotes The amount of HIV-1 gag p24 in concentrated vector stocks was determined by ELISA (see below).
T99839 40659-40753 Sentence denotes The amount of HIV-1 gag p24 in concentrated vector stocks was determined by ELISA (see below).
TextSentencer_T245 40754-40969 Sentence denotes To generate CEMss-CCR5 cells, 1 × 10 5 CEMss cells and 2 × 10 6 TU of recombinant lentiviral vector expressing human CCR5 were added onto 24-well tissue culture plate in the presence of 8 μg/ml of polybrene (Sigma).
T19296 40754-40969 Sentence denotes To generate CEMss-CCR5 cells, 1 × 10 5 CEMss cells and 2 × 10 6 TU of recombinant lentiviral vector expressing human CCR5 were added onto 24-well tissue culture plate in the presence of 8 μg/ml of polybrene (Sigma).
TextSentencer_T246 40970-41054 Sentence denotes Twenty four hours later cells were extensively washed and cultured in complete DMEM.
T29173 40970-41054 Sentence denotes Twenty four hours later cells were extensively washed and cultured in complete DMEM.
TextSentencer_T247 41055-41108 Sentence denotes The expression of CCR5 was measured by FACS analyses.
T4214 41055-41108 Sentence denotes The expression of CCR5 was measured by FACS analyses.
TextSentencer_T248 41109-41236 Sentence denotes Generation of soluble CD4 (sCD4) by drosophila S2 cells Soluble CD4 were generated according to the manufacturer's instruction.
T24924 41109-41236 Sentence denotes Generation of soluble CD4 (sCD4) by drosophila S2 cells Soluble CD4 were generated according to the manufacturer's instruction.
TextSentencer_T249 41237-41340 Sentence denotes Briefly, 2 × 10 6 S2 cells were seeded onto each well of 6-well plate in 3 ml Express FIVE® SFM medium.
T61772 41237-41340 Sentence denotes Briefly, 2 × 10 6 S2 cells were seeded onto each well of 6-well plate in 3 ml Express FIVE® SFM medium.
TextSentencer_T250 41341-41507 Sentence denotes After overnight culture, cells were cotransfected with 19 μg pMT-sCD4 and 1 μg pCoBlast (Invitrogen life Technologies) using a calcium phosphate precipitation method.
T57201 41341-41507 Sentence denotes After overnight culture, cells were cotransfected with 19 μg pMT-sCD4 and 1 μg pCoBlast (Invitrogen life Technologies) using a calcium phosphate precipitation method.
TextSentencer_T251 41508-41688 Sentence denotes Forty eight hours later, 25 μg/ml blasticidin were added into medium to select stably cotransfected cell lines and followed by limiting dilution assay to select stable cell clones.
T94655 41508-41688 Sentence denotes Forty eight hours later, 25 μg/ml blasticidin were added into medium to select stably cotransfected cell lines and followed by limiting dilution assay to select stable cell clones.
TextSentencer_T252 41689-41769 Sentence denotes To produce sCD4, stable clones were induced with 5 μg/ml CdCl 2 for 3 to 5 days.
T20624 41689-41769 Sentence denotes To produce sCD4, stable clones were induced with 5 μg/ml CdCl 2 for 3 to 5 days.
TextSentencer_T253 41770-41896 Sentence denotes Soluble CD4 in supernatants were harvested and purified by Ni-NTA column (Invitrogen) according to manufacturer's instruction.
T27492 41770-41896 Sentence denotes Soluble CD4 in supernatants were harvested and purified by Ni-NTA column (Invitrogen) according to manufacturer's instruction.
TextSentencer_T254 41897-42051 Sentence denotes The amount of proteins was quantified by BCA assay (Pierce) and the purity of proteins was determined by 12% SDS/PAGE followed by Coomassie blue staining.
T32754 41897-42051 Sentence denotes The amount of proteins was quantified by BCA assay (Pierce) and the purity of proteins was determined by 12% SDS/PAGE followed by Coomassie blue staining.
TextSentencer_T255 42052-42362 Sentence denotes To transduce CEMss and CEMss-CCR5 cells, 1 × 10 5 CEMss or CEMss-CCR5 cells and 2 × 10 6 TU of one of the lentiviral vectors containing pRRL-scFv (X5 or AB65)/hinge/his-tag/DAF or pRRL-scFv(X5 or AB65)/ hinge/his-tag were added onto 24-well tissue culture plate in the presence of 8 μg/ml of polybrene (Sigma).
T33873 42052-42362 Sentence denotes To transduce CEMss and CEMss-CCR5 cells, 1 × 10 5 CEMss or CEMss-CCR5 cells and 2 × 10 6 TU of one of the lentiviral vectors containing pRRL-scFv (X5 or AB65)/hinge/his-tag/DAF or pRRL-scFv(X5 or AB65)/ hinge/his-tag were added onto 24-well tissue culture plate in the presence of 8 μg/ml of polybrene (Sigma).
TextSentencer_T256 42363-42447 Sentence denotes Twenty four hours later cells were extensively washed and cultured in complete DMEM.
T65174 42363-42447 Sentence denotes Twenty four hours later cells were extensively washed and cultured in complete DMEM.
TextSentencer_T257 42448-42537 Sentence denotes To transduce TZM.bl cells, 5 × 10 4 TMZ.bl cells per well were seeded onto 24 well plate.
T92109 42448-42537 Sentence denotes To transduce TZM.bl cells, 5 × 10 4 TMZ.bl cells per well were seeded onto 24 well plate.
TextSentencer_T258 42538-42839 Sentence denotes After overnight culture, 2 × 10 6 TU of one of lentiviral vectors containing pRRL-scFv (TG15, AB31, AB32, 4E10, X5, 48d or AB65)/hinge/his-tag/DAF or pRRL-scFv (TG15, AB31, AB32, 4E10, X5, 48d or AB65)/hinge/his-tag were added onto 24-well tissue culture plate in the presence of 8 μg/ml of polybrene.
T58458 42538-42839 Sentence denotes After overnight culture, 2 × 10 6 TU of one of lentiviral vectors containing pRRL-scFv (TG15, AB31, AB32, 4E10, X5, 48d or AB65)/hinge/his-tag/DAF or pRRL-scFv (TG15, AB31, AB32, 4E10, X5, 48d or AB65)/hinge/his-tag were added onto 24-well tissue culture plate in the presence of 8 μg/ml of polybrene.
TextSentencer_T259 42840-42924 Sentence denotes Twenty four hours later cells were extensively washed and cultured in complete DMEM.
T92572 42840-42924 Sentence denotes Twenty four hours later cells were extensively washed and cultured in complete DMEM.
TextSentencer_T260 42925-43109 Sentence denotes Expression of scFv/ hinge/his-tag/DAF constructs was measured by FACS and western blot analyses and expression of scFv/hinge/ his-tag was measured by western blot analysis (see below).
T6869 42925-43109 Sentence denotes Expression of scFv/ hinge/his-tag/DAF constructs was measured by FACS and western blot analyses and expression of scFv/hinge/ his-tag was measured by western blot analysis (see below).
TextSentencer_T261 43110-43218 Sentence denotes We usually found that after a single round transduction, over 98% cells express transgenes (data not shown).
T44162 43110-43218 Sentence denotes We usually found that after a single round transduction, over 98% cells express transgenes (data not shown).
TextSentencer_T262 43219-43336 Sentence denotes After transduced cells were generated, cells were continuously cultured in complete DMEM and split every 3 or 4 days.
T31288 43219-43336 Sentence denotes After transduced cells were generated, cells were continuously cultured in complete DMEM and split every 3 or 4 days.
TextSentencer_T263 43337-43389 Sentence denotes Periodically, expression of transgenes was measured.
T62237 43337-43389 Sentence denotes Periodically, expression of transgenes was measured.
TextSentencer_T264 43390-43500 Sentence denotes We found that the level of transgene expression was very stable in the transduced cell lines (data not shown).
T97065 43390-43500 Sentence denotes We found that the level of transgene expression was very stable in the transduced cell lines (data not shown).
TextSentencer_T265 43501-43823 Sentence denotes To study cell surface expression of scFv/hinge/his-tag/ DAF, 2 × 10 5 mock-and scFv (TG15, AB31, AB32, 4E10, X5, 48d and AB65)/hinge/his-tag/DAF-transduced TZM. bl cells, mock-and scFv (X5 and AB65)/hinge/his-tag/ DAF-transduced CEMss-CCR5 cells were incubated with a mouse anti-his-tag antibody (Sigma) for 45 min on ice.
T59491 43501-43823 Sentence denotes To study cell surface expression of scFv/hinge/his-tag/ DAF, 2 × 10 5 mock-and scFv (TG15, AB31, AB32, 4E10, X5, 48d and AB65)/hinge/his-tag/DAF-transduced TZM. bl cells, mock-and scFv (X5 and AB65)/hinge/his-tag/ DAF-transduced CEMss-CCR5 cells were incubated with a mouse anti-his-tag antibody (Sigma) for 45 min on ice.
TextSentencer_T266 43824-44005 Sentence denotes Cells then were washed twice with FACS buffer (PBS containing 1% BSA and 0.02% NaN 3 ) and stained with PE-conjugated goat anti-mouse IgG antibody (Sigma) for another 45 min on ice.
T83909 43824-44005 Sentence denotes Cells then were washed twice with FACS buffer (PBS containing 1% BSA and 0.02% NaN 3 ) and stained with PE-conjugated goat anti-mouse IgG antibody (Sigma) for another 45 min on ice.
TextSentencer_T267 44006-44108 Sentence denotes Cells then were washed twice with FACS buffer and fixed with 1% formaldehyde in 0.5 ml of FACS buffer.
T45658 44006-44108 Sentence denotes Cells then were washed twice with FACS buffer and fixed with 1% formaldehyde in 0.5 ml of FACS buffer.
TextSentencer_T268 44109-44188 Sentence denotes FACS analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA).
T14780 44109-44188 Sentence denotes FACS analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA).
TextSentencer_T269 44189-44529 Sentence denotes To determine whether the expression of scFv (TG15, AB31, AB32, X5, 48d and AB65)/hinge/his-tag/DAF is truly through a GPI anchor, 2 × 10 5 mock-and scFv/ hinge/his-tag/DAF-transduced TMZ.bl and CEMss-CCR5 cells were first incubated with or without 3 μg/ml PI-PLC (Prozyme, San Leandro, CA, USA) in DMEM containing 1% FBS at 37°C for 30 min.
T86324 44189-44529 Sentence denotes To determine whether the expression of scFv (TG15, AB31, AB32, X5, 48d and AB65)/hinge/his-tag/DAF is truly through a GPI anchor, 2 × 10 5 mock-and scFv/ hinge/his-tag/DAF-transduced TMZ.bl and CEMss-CCR5 cells were first incubated with or without 3 μg/ml PI-PLC (Prozyme, San Leandro, CA, USA) in DMEM containing 1% FBS at 37°C for 30 min.
TextSentencer_T270 44530-44674 Sentence denotes After the incubation, cells were washed twice to remove remaining PI-PLC and then stained with a mouse anti-his-tag antibody as described above.
T38639 44530-44674 Sentence denotes After the incubation, cells were washed twice to remove remaining PI-PLC and then stained with a mouse anti-his-tag antibody as described above.
TextSentencer_T271 44675-44951 Sentence denotes Western blot analysis 1×10 6 cells [TZM.bl-GPI-scFvs (AB65, TG15, AB31, AB32, X5 and 48d), TZM.bl-scFvs (AB65, TG15, AB31, AB32, X5 and 48d), CEMss-R5-GPI-scFvs(AB65 and X5) and CEMss-R5-scFvs(AB65 and X5)] were grown in the DMEM plus 1% FBS on the 12-well plate for 24 hours.
T10835 44675-44951 Sentence denotes Western blot analysis 1×10 6 cells [TZM.bl-GPI-scFvs (AB65, TG15, AB31, AB32, X5 and 48d), TZM.bl-scFvs (AB65, TG15, AB31, AB32, X5 and 48d), CEMss-R5-GPI-scFvs(AB65 and X5) and CEMss-R5-scFvs(AB65 and X5)] were grown in the DMEM plus 1% FBS on the 12-well plate for 24 hours.
TextSentencer_T272 44952-45002 Sentence denotes Cells and supernatant were harvested respectively.
T67403 44952-45002 Sentence denotes Cells and supernatant were harvested respectively.
TextSentencer_T273 45003-45130 Sentence denotes Cells were lysed by lysis buffer (100 mM Tris-HCl, pH8.0, 1% NP40) in the presence of protease inhibitor cocktail (Calbiochem).
T95010 45003-45130 Sentence denotes Cells were lysed by lysis buffer (100 mM Tris-HCl, pH8.0, 1% NP40) in the presence of protease inhibitor cocktail (Calbiochem).
TextSentencer_T274 45131-45256 Sentence denotes Proteins in supernatant were precipitated by TCA and dissolved in the equal volume of lysis buffer that to lysed cell pellet.
T909 45131-45256 Sentence denotes Proteins in supernatant were precipitated by TCA and dissolved in the equal volume of lysis buffer that to lysed cell pellet.
TextSentencer_T275 45257-45384 Sentence denotes Samples were detected by mouse anti-His tag antibody (Sigma) and mouse anti human beta-tubulin antibody as a reference control.
T71431 45257-45384 Sentence denotes Samples were detected by mouse anti-His tag antibody (Sigma) and mouse anti human beta-tubulin antibody as a reference control.
TextSentencer_T276 45385-45769 Sentence denotes To generate pseudotypes with the HIV-1 vector, 4 × 10 6 293T packaging cells were co-transfected with 10 μg of HIV-1-luciferase transfer vector [38] and 10 μg of DNA plasmid encoding one of HIV-1 envelopes (Q168, AD8, ADA, JR-FL, Yu2, HxBc2, consensus B, Mj4, CN054, CNE8, CNE11 or CNE17) or control retroviral envelope 10A1 using a calcium phosphate precipitation method (see above).
T85385 45385-45769 Sentence denotes To generate pseudotypes with the HIV-1 vector, 4 × 10 6 293T packaging cells were co-transfected with 10 μg of HIV-1-luciferase transfer vector [38] and 10 μg of DNA plasmid encoding one of HIV-1 envelopes (Q168, AD8, ADA, JR-FL, Yu2, HxBc2, consensus B, Mj4, CN054, CNE8, CNE11 or CNE17) or control retroviral envelope 10A1 using a calcium phosphate precipitation method (see above).
TextSentencer_T277 45770-45870 Sentence denotes DNA plasmids encoding HIV-1 envelopes Q168, AD8, ADA, JR-FL, HxBc2 and Mj4 were obtained from ARRRP.
T46081 45770-45870 Sentence denotes DNA plasmids encoding HIV-1 envelopes Q168, AD8, ADA, JR-FL, HxBc2 and Mj4 were obtained from ARRRP.
TextSentencer_T278 45871-45930 Sentence denotes DNA plasmid encoding consensus B was a generous gift of Dr.
T48099 45871-45930 Sentence denotes DNA plasmid encoding consensus B was a generous gift of Dr.
TextSentencer_T279 45931-45935 Sentence denotes B.H.
T37945 45931-45935 Sentence denotes B.H.
TextSentencer_T280 45936-45970 Sentence denotes Hahn at the University of Alabama.
T29771 45936-45970 Sentence denotes Hahn at the University of Alabama.
TextSentencer_T281 45971-46076 Sentence denotes DNA plasmids encoding CNE8, CNE11 or CNE17 were obtained from Dr. Linqi Zhang at the Tsinghua University.
T5497 45971-46076 Sentence denotes DNA plasmids encoding CNE8, CNE11 or CNE17 were obtained from Dr. Linqi Zhang at the Tsinghua University.
TextSentencer_T282 46077-46145 Sentence denotes HIV-1 envelope Q168 is derived from a R5 tropic clade A virus [63] .
T41102 46077-46145 Sentence denotes HIV-1 envelope Q168 is derived from a R5 tropic clade A virus [63] .
TextSentencer_T283 46146-46247 Sentence denotes HIV-1 envelopes AD8, ADA, JR-FL, Yu2 are derived from R5 tropic clade B viruses [64] [65] [66] [67] .
T49577 46146-46247 Sentence denotes HIV-1 envelopes AD8, ADA, JR-FL, Yu2 are derived from R5 tropic clade B viruses [64] [65] [66] [67] .
TextSentencer_T284 46248-46317 Sentence denotes HIV-1 envelope HxBc2 is derived from a X4 tropic clade B virus [68] .
T84270 46248-46317 Sentence denotes HIV-1 envelope HxBc2 is derived from a X4 tropic clade B virus [68] .
TextSentencer_T285 46318-46423 Sentence denotes HIV-1 envelope consensus B is an artificial envelope originally generated in Dr. Hahn's laboratory [69] .
T21024 46318-46423 Sentence denotes HIV-1 envelope consensus B is an artificial envelope originally generated in Dr. Hahn's laboratory [69] .
TextSentencer_T286 46424-46492 Sentence denotes HIV-1 envelopes Mj4 is derived from R5 tropic clade C viruses [70] .
T23319 46424-46492 Sentence denotes HIV-1 envelopes Mj4 is derived from R5 tropic clade C viruses [70] .
TextSentencer_T287 46493-46562 Sentence denotes HIV-1 envelope CNE8 is derived from a R5 tropic clade CRF01_AE virus.
T37660 46493-46562 Sentence denotes HIV-1 envelope CNE8 is derived from a R5 tropic clade CRF01_AE virus.
TextSentencer_T288 46563-46627 Sentence denotes HIV-1 envelope CNE11 is derived from a R5 tropic clade B' virus.
T96508 46563-46627 Sentence denotes HIV-1 envelope CNE11 is derived from a R5 tropic clade B' virus.
TextSentencer_T289 46628-46732 Sentence denotes HIV-1 envelope CNE17 is derived from a R5 tropic clade CRF07_B'C virus (Zhang et al. unpublished data) .
T46219 46628-46732 Sentence denotes HIV-1 envelope CNE17 is derived from a R5 tropic clade CRF07_B'C virus (Zhang et al. unpublished data) .
TextSentencer_T290 46733-46834 Sentence denotes The pseudotype-containing supernatants were harvested and stored in aliquots at -80°C degree freezer.
T46659 46733-46834 Sentence denotes The pseudotype-containing supernatants were harvested and stored in aliquots at -80°C degree freezer.
TextSentencer_T291 46835-46907 Sentence denotes The amount of HIV-1 p24 in collected supernatants was measured by ELISA.
T13435 46835-46907 Sentence denotes The amount of HIV-1 p24 in collected supernatants was measured by ELISA.
TextSentencer_T292 46908-47194 Sentence denotes In a single-cycle assay to measure the infectivity of pseudotypes, 10,000 Mock-, scFv/hinge/his-tag-, scFv/hinge/ his-tag/DAF-transduced TZM.bl cells were transduced with HIV-1 pseudotype-containing supernatants equivalent to relative luciferase activity 300,000 to 1,000,000 overnight.
T64442 46908-47194 Sentence denotes In a single-cycle assay to measure the infectivity of pseudotypes, 10,000 Mock-, scFv/hinge/his-tag-, scFv/hinge/ his-tag/DAF-transduced TZM.bl cells were transduced with HIV-1 pseudotype-containing supernatants equivalent to relative luciferase activity 300,000 to 1,000,000 overnight.
TextSentencer_T293 47195-47281 Sentence denotes Cells were then washed twice with PBS and cultured in complete DMEM medium for 2 days.
T44555 47195-47281 Sentence denotes Cells were then washed twice with PBS and cultured in complete DMEM medium for 2 days.
TextSentencer_T294 47282-47355 Sentence denotes Cells were then washed once with PBS and lysed in 100 μl of lysis buffer.
T47365 47282-47355 Sentence denotes Cells were then washed once with PBS and lysed in 100 μl of lysis buffer.
TextSentencer_T295 47356-47504 Sentence denotes Luciferase activity in 50 μl of cell suspensions was measured by a BrightGlo Luciferase assay according to the manufacturer's instruction (Promega).
T78023 47356-47504 Sentence denotes Luciferase activity in 50 μl of cell suspensions was measured by a BrightGlo Luciferase assay according to the manufacturer's instruction (Promega).
TextSentencer_T296 47505-47634 Sentence denotes Mock-, scFv/hinge/his-tag-, scFv/hinge/his-tag/DAFtransduced TMZ.bl cells at 5,000 cells per well were seeded onto 96-well plate.
T40897 47505-47634 Sentence denotes Mock-, scFv/hinge/his-tag-, scFv/hinge/his-tag/DAFtransduced TMZ.bl cells at 5,000 cells per well were seeded onto 96-well plate.
TextSentencer_T297 47635-47806 Sentence denotes After the overnight culture, cells were infected with HIV-1 strains Bru-3, Bru-YU2, AD8, Mj4, RJ407 and JS-JCD (100 to 200 TCID 50 ) in a final volume of 0.5 ml overnight.
T46390 47635-47806 Sentence denotes After the overnight culture, cells were infected with HIV-1 strains Bru-3, Bru-YU2, AD8, Mj4, RJ407 and JS-JCD (100 to 200 TCID 50 ) in a final volume of 0.5 ml overnight.
TextSentencer_T298 47807-47937 Sentence denotes Cells were then washed three times with HBSS and resuspended in 2 ml of the complete DMEM medium and incubated at 37°C for 2 days.
T62702 47807-47937 Sentence denotes Cells were then washed three times with HBSS and resuspended in 2 ml of the complete DMEM medium and incubated at 37°C for 2 days.
TextSentencer_T299 47938-48018 Sentence denotes Infectivity of HIV-1 was determined by a BrightGlo Luciferase assay (see above).
T27777 47938-48018 Sentence denotes Infectivity of HIV-1 was determined by a BrightGlo Luciferase assay (see above).
TextSentencer_T300 48019-48252 Sentence denotes To test the effect of sCD4 on anti-HIV potency of secretory and GPI-anchored scFvs (X5), parental TZM. bl cells, TZM.bl-scFv (X5) and TZM.bl-GPI-scFv (X5) (5,000 cells per well) were seeded onto 96-well plate and incubated overnight.
T69848 48019-48252 Sentence denotes To test the effect of sCD4 on anti-HIV potency of secretory and GPI-anchored scFvs (X5), parental TZM. bl cells, TZM.bl-scFv (X5) and TZM.bl-GPI-scFv (X5) (5,000 cells per well) were seeded onto 96-well plate and incubated overnight.
TextSentencer_T301 48253-48399 Sentence denotes 400 TCID 50 and 4,000 TCID 50 of HIV-1 Bru-3 and Bru-Yu2 were first mixed with or without pre-determined 0.3 μg/ml of sCD4 at 37°C for 15 minutes.
T14960 48253-48399 Sentence denotes 400 TCID 50 and 4,000 TCID 50 of HIV-1 Bru-3 and Bru-Yu2 were first mixed with or without pre-determined 0.3 μg/ml of sCD4 at 37°C for 15 minutes.
TextSentencer_T302 48400-48521 Sentence denotes Viruses with or without sCD4 were then added onto parental TZM.bl cells, TZM.bl-scFv (X5) and TZM.bl-GPI-scFv (X5) cells.
T55659 48400-48521 Sentence denotes Viruses with or without sCD4 were then added onto parental TZM.bl cells, TZM.bl-scFv (X5) and TZM.bl-GPI-scFv (X5) cells.
TextSentencer_T303 48522-48614 Sentence denotes After 2 days, infectivity of HIV-1 was determined by BrightGlo Luciferase assay (see above).
T36847 48522-48614 Sentence denotes After 2 days, infectivity of HIV-1 was determined by BrightGlo Luciferase assay (see above).
TextSentencer_T304 48615-48857 Sentence denotes Long term culture of HIV-1 infected cells 1×10 6 CEMss-CCR5 cells transduced with GPI-scFvs (X5 or AB65) and secretory scFvs (X5 or AB65) were infected with HIV-1 strains Bru-3 and Bru-Yu2 (200 TCID 50 ) in a final volume of 0.5 ml overnight.
T79601 48615-48857 Sentence denotes Long term culture of HIV-1 infected cells 1×10 6 CEMss-CCR5 cells transduced with GPI-scFvs (X5 or AB65) and secretory scFvs (X5 or AB65) were infected with HIV-1 strains Bru-3 and Bru-Yu2 (200 TCID 50 ) in a final volume of 0.5 ml overnight.
TextSentencer_T305 48858-48972 Sentence denotes Cells were then extensively washed with HBSS, resuspended in 6 ml of complete DMEM and cultured for 3 to 15 weeks.
T32068 48858-48972 Sentence denotes Cells were then extensively washed with HBSS, resuspended in 6 ml of complete DMEM and cultured for 3 to 15 weeks.
TextSentencer_T306 48973-49064 Sentence denotes Every 3 days, 4.5 ml of cell suspensions were harvested and replaced with the fresh medium.
T57351 48973-49064 Sentence denotes Every 3 days, 4.5 ml of cell suspensions were harvested and replaced with the fresh medium.
TextSentencer_T307 49065-49102 Sentence denotes The supernatants were then collected.
T57576 49065-49102 Sentence denotes The supernatants were then collected.
TextSentencer_T308 49103-49218 Sentence denotes HIV-1 p24 in the supernatants were measured by ELISA (Beckman Coulter) according to the manufacturer's instruction.
T36510 49103-49218 Sentence denotes HIV-1 p24 in the supernatants were measured by ELISA (Beckman Coulter) according to the manufacturer's instruction.
TextSentencer_T309 49219-49376 Sentence denotes Cell-cell fusion experiment 69TiRev/Env cells (1×10 5 per well) with or without 2 μg/ml tetracycline treatment were grown on 12-well plate at 37°C overnight.
T62475 49219-49376 Sentence denotes Cell-cell fusion experiment 69TiRev/Env cells (1×10 5 per well) with or without 2 μg/ml tetracycline treatment were grown on 12-well plate at 37°C overnight.
TextSentencer_T310 49377-49455 Sentence denotes CEMss-GPI-scFvs (AB65 or X5) (2×10 5 ) were then added onto 69TiRev/Env cells.
T15077 49377-49455 Sentence denotes CEMss-GPI-scFvs (AB65 or X5) (2×10 5 ) were then added onto 69TiRev/Env cells.
TextSentencer_T311 49456-49539 Sentence denotes Cellcell fusion was monitored under the light microscopy at various time intervals.
T92645 49456-49539 Sentence denotes Cellcell fusion was monitored under the light microscopy at various time intervals.
TextSentencer_T312 49540-49649 Sentence denotes When fusion process reached peak at 20 hours, the results were recorded by CCD digital camera (Leica DM IRB).
T19083 49540-49649 Sentence denotes When fusion process reached peak at 20 hours, the results were recorded by CCD digital camera (Leica DM IRB).
TextSentencer_T313 49650-49756 Sentence denotes Peripheral blood was obtained from healthy donors at the Gulf Coast Regional Blood Center, Houston, Texas.
T13011 49650-49756 Sentence denotes Peripheral blood was obtained from healthy donors at the Gulf Coast Regional Blood Center, Houston, Texas.
TextSentencer_T314 49757-49885 Sentence denotes Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation as described before [71] .
T65036 49757-49885 Sentence denotes Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation as described before [71] .
TextSentencer_T315 49886-50042 Sentence denotes To isolate CD14 + monocytes, the anti-CD14 microbeads and miniMACS system were used according to manufacturer's instructions (Miltenyi Biotech, Auburn, CA).
T1024 49886-50042 Sentence denotes To isolate CD14 + monocytes, the anti-CD14 microbeads and miniMACS system were used according to manufacturer's instructions (Miltenyi Biotech, Auburn, CA).
TextSentencer_T316 50043-50148 Sentence denotes Briefly, 10 8 PBMCs were resuspended in 800 μl of binding buffer (PBS containing 0.5% BSA and 2 mM EDTA).
T70857 50043-50148 Sentence denotes Briefly, 10 8 PBMCs were resuspended in 800 μl of binding buffer (PBS containing 0.5% BSA and 2 mM EDTA).
TextSentencer_T317 50149-50234 Sentence denotes 200 μl of MACS CD14 microbeads were added, mixed and incubated at 4°C for 15 minutes.
T52450 50149-50234 Sentence denotes 200 μl of MACS CD14 microbeads were added, mixed and incubated at 4°C for 15 minutes.
TextSentencer_T318 50235-50346 Sentence denotes The cell-bead mixture was then washed once with binding buffer and resuspended in 1 ml of fresh binding buffer.
T31887 50235-50346 Sentence denotes The cell-bead mixture was then washed once with binding buffer and resuspended in 1 ml of fresh binding buffer.
TextSentencer_T319 50347-50414 Sentence denotes CD14 + cells were positively selected using an MS+/RS + column tip.
T90726 50347-50414 Sentence denotes CD14 + cells were positively selected using an MS+/RS + column tip.
TextSentencer_T320 50415-50657 Sentence denotes The isolated cells were washed once with complete RPMI and cultured in the complete RPMI supplemented with GM-CSF (1000 U/ ml) and IL-4 (1000 U/ml) (R & D System, Minneapolis, MN) for 7 days to generate immature DCs as described before [72] .
T66267 50415-50657 Sentence denotes The isolated cells were washed once with complete RPMI and cultured in the complete RPMI supplemented with GM-CSF (1000 U/ ml) and IL-4 (1000 U/ml) (R & D System, Minneapolis, MN) for 7 days to generate immature DCs as described before [72] .
TextSentencer_T321 50658-50819 Sentence denotes The expression of DC-SIGN on the cell surface of immature DCs was measured by anti-DC-SIGN antibody staining followed by FACS analysis as described before [23] .
T53084 50658-50819 Sentence denotes The expression of DC-SIGN on the cell surface of immature DCs was measured by anti-DC-SIGN antibody staining followed by FACS analysis as described before [23] .
TextSentencer_T322 50820-50995 Sentence denotes DCs (10 5 per well) were seeded in triplicates in a 96 well U-bottom plate and incubated with 100 TCID 50 of NL4-3 in a total volume of 200 μl at 37°C and 5% CO 2 for 4 hours.
T70372 50820-50995 Sentence denotes DCs (10 5 per well) were seeded in triplicates in a 96 well U-bottom plate and incubated with 100 TCID 50 of NL4-3 in a total volume of 200 μl at 37°C and 5% CO 2 for 4 hours.
TextSentencer_T323 50996-51205 Sentence denotes Cells were then washed three times with PBS to remove cell-free virus and resuspended in 2 ml of complete RPMI into wells of 24-well plate with or without adding 1.5 × 10 5 CEMss-GPI-scFvs (AB65 and X5) cells.
T12973 50996-51205 Sentence denotes Cells were then washed three times with PBS to remove cell-free virus and resuspended in 2 ml of complete RPMI into wells of 24-well plate with or without adding 1.5 × 10 5 CEMss-GPI-scFvs (AB65 and X5) cells.
TextSentencer_T324 51206-51254 Sentence denotes Cells were continued to be cultured for 14 days.
T68726 51206-51254 Sentence denotes Cells were continued to be cultured for 14 days.
TextSentencer_T325 51255-51348 Sentence denotes Every 3 or 4 days, medium was removed from each well and replaced with fresh complete medium.
T84361 51255-51348 Sentence denotes Every 3 or 4 days, medium was removed from each well and replaced with fresh complete medium.
TextSentencer_T326 51349-51420 Sentence denotes HIV-1 p24 in the supernatants was measured by ELISA as described above.
T96019 51349-51420 Sentence denotes HIV-1 p24 in the supernatants was measured by ELISA as described above.
TextSentencer_T327 51421-51620 Sentence denotes Parental and TZM.bl-GPI-scFvs (AB65 and X5) cells were seeded (5,000 cells per well) onto the Lab-Tek chamber slide (Nalge Nunc International, Rochester, NY) and incubated at 37°C 5% CO 2 for 2 days.
T68104 51421-51620 Sentence denotes Parental and TZM.bl-GPI-scFvs (AB65 and X5) cells were seeded (5,000 cells per well) onto the Lab-Tek chamber slide (Nalge Nunc International, Rochester, NY) and incubated at 37°C 5% CO 2 for 2 days.
TextSentencer_T328 51621-51742 Sentence denotes Cells were then washed twice with 500 μl PBS and fixed by fixation buffer (4% formaldehyde in PBS plus 1%BSA) for 15 min.
T18690 51621-51742 Sentence denotes Cells were then washed twice with 500 μl PBS and fixed by fixation buffer (4% formaldehyde in PBS plus 1%BSA) for 15 min.
TextSentencer_T329 51743-51861 Sentence denotes Cells were washed twice with 500 μl PBS and blocked with blockage buffer (5% goat serum in PBS plus 1%BSA) for 1 hour.
T3121 51743-51861 Sentence denotes Cells were washed twice with 500 μl PBS and blocked with blockage buffer (5% goat serum in PBS plus 1%BSA) for 1 hour.
TextSentencer_T330 51862-51961 Sentence denotes Cells were stained with Alexa 555 conjugated CtxB (Invitrogen Life Technologies) at 4°C for 45 min.
T62791 51862-51961 Sentence denotes Cells were stained with Alexa 555 conjugated CtxB (Invitrogen Life Technologies) at 4°C for 45 min.
TextSentencer_T331 51962-52155 Sentence denotes After washed 3 times with PBS, cells stained with mouse anti-his-tag antibody (Sigma) at 4°C for 45 min, then stained with Alexa 488-conjugated goat anti-mouse IgG antibody (Invitrogen) at 4°C.
T49574 51962-52155 Sentence denotes After washed 3 times with PBS, cells stained with mouse anti-his-tag antibody (Sigma) at 4°C for 45 min, then stained with Alexa 488-conjugated goat anti-mouse IgG antibody (Invitrogen) at 4°C.
TextSentencer_T332 52156-52331 Sentence denotes After cells were washed 3 times with PBS, cells were stained with 4',6-diamidino-2phenylindole (DAPI) in permeabilization buffer (blockage buffer plus 0.5% saponin) for 5 min.
T15355 52156-52331 Sentence denotes After cells were washed 3 times with PBS, cells were stained with 4',6-diamidino-2phenylindole (DAPI) in permeabilization buffer (blockage buffer plus 0.5% saponin) for 5 min.
TextSentencer_T333 52332-52438 Sentence denotes The slides were mounted before being analyzed under confocal fluorescent microscope (Zeiss Model LSM 510).
T4859 52332-52438 Sentence denotes The slides were mounted before being analyzed under confocal fluorescent microscope (Zeiss Model LSM 510).