Department of Biological Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba 278-8510, Japan1
Department of Pathology, National Institute for Infectious Diseases, Tokyo 162-8640, Japan2
Author for correspondence: Joe Chiba. Fax +81 471 25 1841. e-mail chibaj{at}rs.noda.sut.ac.jp
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Abstract |
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Introduction |
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Residues 259 to 284 in the thumb subdomain exhibit sequence homology with other nucleic acid polymerases and have been termed the helix clump. This amino acid motif has been identified in the crystal structure model as an element of the enzymes nucleic acid-binding apparatus (Hermann et al., 1994 ). Analyses using RT mutants containing alanine substitutions in the helix clump suggest that the alpha H core (residues Gln258, Gly262 and Trp266) interacts with the template primer (Beard et al., 1994
). Interactions between specific amino acids and the primer stem at positions well removed from the active site are critical determinants of processivity and fidelity (Bebenek et al., 1995
). Recently, residues in the thumb subdomain and the minor groove-binding track, in particular, have been reported to be crucial for unique interactions between RT and the polypurine tract, which is required for correct positioning and precise RNase H cleavage (Powell et al., 1999
).
Our previous study revealed the existence of a specific neutralizing epitope that is recognized by a murine monoclonal antibody (MAb), 7C4, at a position close to, or forming part of, this highly conserved region of HIV-1 RT. MAb 7C4 interferes with the interaction between RT and the template primer and strongly inhibits the RNA-dependent polymerase activity of HIV-1 RT (Chiba et al., 1996 ). Among the various retrovirus polymerases, 7C4 seems to be specific for HIV-1 RT: 7C4 inhibited the RT activity of three strains of HIV-1 (IIIB, Bru and IMS-1), but did not inhibit the RT activity of two strains of either HIV-2 (GH-1 and LAV-2) or simian immunodeficiency virus (MAC and MND) (Chiba et al., 1997
). If additional MAbs similar to this unique antibody were available they might provide information about the function of the helix clump of HIV-1 RT and assist in the analyses of RT mutants containing alanine substitutions in the helix clump.
This study was initially aimed at developing a panel of recombinant MAbs that are reactive with various epitopes on HIV-1 RT using a phage display library method. In the course of this study, we have succeeded in producing two additional MAbs reactive with an epitope which is probably the same as, or located close to, the 7C4 epitope and is specific for HIV-1 RT among various DNA polymerases. We designate this epitope HRSINE by capitalizing the first letters of HIV-1 RT-specific immunodominant and neutralization epitope. HRSINE is a logical target for the development of new types of HIV-1 RT inhibitors. Moreover, these recombinant antibody fragments, 5F, 5G and 7C4, may themselves serve as strong and specific inhibitors of HIV-1 replication when expressed intracellularly in human cells as intrabodies or intracellular antibodies. Here, we describe highly efficient cloning of cDNA encoding Fab fragments that react with HRSINE by combining the phage display library method with an immunization method that uses an infectious vaccinia virus recombinant.
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Methods |
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Construction of the phage display library.
We constructed a phage display library of immunoglobulin cDNA from the spleen cells of immunized mice using a pComb3 vector system (Barbas & Lerner, 1991 ; Burton et al., 1991
) with slight modification as follows. The phagemid expression vector for the library was pComb3H (Rader & Barbas, 1997
). Synthetic DNA encoding a histidine hexamer followed by a termination codon was cloned into pComb3H between the NheI and NotI sites to enable purification of expressed recombinant Fab fragments. The conditions for amplification of immunoglobulin cDNA with each combination of the PCR primers were optimized by varying pH from 8·5 to 10·0 and MgCl2 concentrations from 1·5 to 3·5 mM. Prior to infection of Escherichia coli XL1-Blue cells with the helper phage VCS-M13, cells were grown in SB medium containing 10 µg/ml tetracycline and 5 mM glucose to abort expression of the Fab fragments. Glucose, tetracycline and uninfected helper phage were removed from the culture by centrifugation 2 h after incubation with helper phage. Cells were then suspended in SB medium containing 20 µg/ml ampicillin and 70 µg/ml kanamycin and incubated at 30 °C overnight. The recombinant phage, precipitated with polyethylene glycol 8000, was resuspended in Dulbeccos PBS containing 10% Block Ace (Dainippon Pharmaceuticals).
Screening for RT-specific Fab fragments.
The recombinant phage library was subjected to affinity selection with immobilized RT (Research Institute for Microbial Diseases, Osaka, Japan). Microtitre plates (Costar 3690) were coated overnight with 50 µl RT per well (20 µg/ml solution in PBS) at 4 °C and blocked with 25% Block Ace in PBS at room temperature for 3 h. Affinity selection followed by amplification of the selected phage was repeated in four rounds. Aliquots of 50 µl phage suspension per well were incubated at 37 °C for 2 h. Wells were then washed with PBS containing 0·5% Tween-20 (PBSTween) once, five and ten times in the first, second and third to fourth rounds of selection, respectively. After four rounds of panning, 20 phage clones were picked to test their antigen-binding activity and for nucleic acid sequencing.
Expression and purification of recombinant Fab fragments.
E. coli cells transformed with the phagemid DNA were cultured at 30 °C overnight in 2 l of SB medium containing 1 mM IPTG. Cells were collected by centrifugation at 1000 g, suspended in 30 ml of ice-cold osmotic solution (30 mM TrisHCl, pH 7·4, containing 20% sucrose) and placed on ice for 1 h. Cells were shocked by the addition of 200 ml of ice-cold water (Sawyer & Blattner, 1991 ). The cell extract was recovered by centrifugation at 10000 g and the salt concentration was adjusted to 500 mM NaCl. The sample was then applied to a column containing 2 ml of Ni-NTA agarose (Qiagen), which had been pre-equilibrated with wash buffer (50 mM TrisHCl, pH 8·0, containing 500 mM NaCl). The column was washed with 9 ml of wash buffer containing 30 mM imidazole and then the bound proteins were eluted with 4 ml of elution buffer (100 mM imidazoleHCl, pH 8·0, containing 500 mM NaCl). The eluted solution was dialysed against 20 mM TrisHCl (pH 8·5) and applied to an anion-exchange column (DEAE-5PW, TOSOH). The column was then washed with 10 ml of the same buffer. Bound protein was eluted with a linear gradient (01 M) of NaCl. Anti-RT antibody activity in each fraction was monitored by ELISA and the active fractions were pooled and dialysed against PBS. Fab fragments were concentrated with Centricon 30 (Amicon) and protein concentration was determined with a BCA protein assay kit (Pierce).
Expression and purification of soluble Fab fragments of MAb 7C4 in E. coli.
The 5' end sequences of the heavy and light chain cDNA from 7C4 hybridoma cells were determined by rapid amplification of the cDNA ends (Frohman et al., 1988 ) using an Fd 3' primer, IgG1 (5' AGGCTTACTAGTACAATCCCTGGGCACAAT 3'), and a
light chain 3' primer (5' GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA 3') (Kang et al., 1991
) as internal primers. DNA sequences encoding the heavy and light chains of MAb 7C4 were then cloned by PCR amplification. Primers for the heavy chain were modified heavy chain variable 5' primer Hc1 (Kang et al., 1991
) with the correct 5' end sequence of the 7C4 heavy chain cDNA (5' AACCAGCCATGGCCGAGGTGCAGCTGGTCGAGTCTGGAGGA 3') and the 3' primer IgG1. Primers for the light chain were modified light chain 5' primer Lc3 (Kang et al., 1991
) with the correct 5' end sequence of the 7C4 light chain cDNA (5' GACGACGGCCCAGGCGGCCCAAATTGTTCTCACCCAGTCT 3') and the
light chain 3' primer. The heavy chain Fd and light chain genes of 7C4 were then successively cloned into the pComb3H vector (Rader & Barbas, 1997
). 7C4 Fab fragments were produced in E. coli cells and purified as described above.
Preparation of recombinant Fab fragments specific for haemocyanin.
We also constructed a phage display library of immunoglobulin cDNA from the spleen cells of a mouse immunized with keyhole limpet haemocyanin (KLH). Fab fragments specific for KLH were prepared as described above.
RT enzymatic activity assay.
RNA-dependent DNA polymerase activity of RT was assayed essentially as described by Hoffman et al. (1985) with slight modification (Chiba et al., 1996
). For determining the inhibitory activity of Fab fragments on RT, 0·25 µg/ml RT was incubated with different concentrations of Fab fragments in 50 mM TrisHCl, pH 7·4, containing 100 mM NaCl, 5 mM EDTA and 0·5 mg/ml BSA at room temperature for 40 min prior to the polymerase reaction.
Epitope mapping of Fab fragments.
To map the epitope recognized by the Fab fragments, their reactivity with various segments of the p66 subunit of HIV-1 RT were determined by Western blotting. The plasmids pRN2161, pRN1161, pRN2022 and pRN4891 (kind gifts from A. Saito and H. Shinagawa, Osaka University, Japan) expressed p66 subunit peptides 5298, 52335, 145428 and 155250, respectively. These peptide segments were expressed in a fused form with -galactosidase in E. coli. Cell lysate prepared with lysozyme was separated by 7·5% SDSPAGE. Proteins were electrophoretically transferred from the gel to a PVDF membrane filter (Millipore), which was subsequently blocked with PBS containing 25% Block Ace and cut into strips. Each strip was incubated for 1 h with 1 ml of 1 µg/ml Fab fragments in PBSTween containing 10% Block Ace. After washing with PBSTween, the strips were incubated with alkaline phosphatase-conjugated goat anti-mouse IgG F(ab')2 (Pierce) diluted to 1/2000 in PBSTween. After three washes, the bands of reactive fusion protein were visualized by incubation with BCIP/NBT substrate (100 mM TrisHCl, pH 9·8, containing 100 mM NaCl, 1·5 mM MgCl2, 0·016% 5-bromo-4-chloro-3-indolyl phosphate, 0·033% nitro blue tetrazolium and 1·46% dimethyl formamide).
Competition ELISA.
Each well of an ELISA plate (Costar 3690) was coated with 50 µl of 1 µg/ml recombinant RT in 50 mM carbonate buffer, pH 9·6, for 3 h. Wells were blocked with PBS containing 25% Block Ace for 1 h. Different concentrations of 50 µl aliquots of competitor Fab fragments diluted with PBSTween containing 10% Block Ace were incubated in the washed wells for 1 h. After washing, 50 µl of 2 µg/ml biotinylated 7C4 IgG or biotinylated 6B9 IgG was added to each well and incubated for 1 h. MAb 6B9 binds to HIV-1 RT without any effect on enzyme activity (Chiba et al., 1996 ) and the 6B9 epitope exists on the palm subdomain (see Fig. 6
). After another wash, 50 µl alkaline phosphatase-labelled streptavidin (GIBCO) diluted to 1/5000 in PBSTween was added to each well and incubated for 1 h. Substrate solution (100 µl) of 1 mg/ml p-nitrophenyl phosphate dissolved in 9·7% diethanolamine, pH 9·8, containing 0·01% MgCl2 and 0·02% NaN3 was added to each well and the amount of bound antibody fragment was measured by absorbance at 405 nm. The background absorbance at 630 nm was subtracted from the A405 values. All procedures were performed at room temperature.
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Sequencing of antibody cDNA.
Nucleic acid sequencing was carried out using the Thermo Sequenase II dye terminator cycle sequencing kit (AmershamPharmacia).
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Results |
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Comparison of amino acid sequences
5F and 5G Fab fragment amino acid sequences were compared with those of the 7C4 Fab fragments (Fig. 6). Interestingly, although the amino acid sequences of the VL region of 5F and 5G Fab were different from that of the same region of the 7C4 Fab, 5F and 5G Fab fragments were almost the same: only one amino acid difference, which was located in the CDR3 region, existed. While the amino acid sequences of the CL region of 5F and 5G Fab fragments were slightly different from that of the 7C4 Fab, 5F and 5G Fab fragments were exactly the same. These results indicate that the cDNAs encoding the light chains of 5F and 5G were probably cloned from B cell clones of the same origin. Surprisingly, the amino acid sequence of the VH region of 5G Fab was nearly the same as that of the 7C4 Fab: only one amino acid difference was seen in the FR3 region but the length and sequence of their CDR3 regions were exactly the same. The amino acid sequence of the VH region of 5F Fab was different from that of the 5G and 7C4 Fab fragments; many amino acids differed throughout this region and a marked difference in the length and sequence in the CDR3 region was noted. These results indicate that the cDNAs encoding the heavy chains of 5F and 5G Fab fragments were cloned from different anti-RT antibody producing B cell clones.
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Discussion |
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We have succeeded in preparing high affinity and enzyme-neutralizing Fab fragments (5F and 5G) directed against HIV-1 RT by the phage display method. The KA values of the 5F and 5G Fab fragments were in the 10-8 M range and are about ten times greater than those of the 7C4 Fab fragments expressed in E. coli (Fig. 6) or those prepared by papain digestion of the 7C4 hybridoma cell-produced protein (data not shown). The 5G and 7C4 Fab fragments showed similar association kinetics and had almost identical amino acid sequences of their heavy chains (Fig. 6
). However, the 7C4 Fab showed lower dissociation kinetics than 5G Fab. Thus, the higher affinity of 5G Fab to that of the 7C4 Fab may be due to the light chain partner of 5G Fab. On the other hand, 5F and 5G Fab fragments showed similar dissociation kinetics and had very similar light chains, although they were combined with different heavy chains. Such different combinations of almost identical heavy and light chains support the idea that each of the 7C4, 5F and 5G Fab fragments recognizes the same epitope with different affinities. Correlation of the neutralizing activity of the 5F, 5G and 7C4 Fab fragments (Fig. 2
) with their affinity for HIV-1 RT (Fig. 6
) and their highly restricted reactivity to the RT of HIV-1 among various other polymerases adds further support to the idea above. Isolation of the 5F and 5G clones from the library might have been a result of random chain shuffling of the cDNA from expanded rare B cell clones encoding light and heavy chains and this might have caused an increase in their affinity.
From the results of amino acid sequence comparison of the 5F, 5G and 7C4 Fab fragments, it was ascertained that expansion of rare B cell clones occurs in mice repeatedly immunized with WRRT. These rare B cell clones probably have immunoglobulin genes encoding the heavy chains of either the 7C4- or the 5F-type and light chains of either the 7C4- or the 5F- and 5G-type or one of the combinations thereof.
To date, it has been difficult to produce enzyme-neutralizing MAbs by the conventional immunization protocol. In both this study and our previous study using mice immunized with WRRT, we report or reported the very efficient production of such MAbs using the phage display method and hybridoma techniques, respectively. Since immunization of mice with WRRT seems to induce the clonal expansion of rare B cells in vivo, we conclude that it is not difficult to induce enzyme-neutralizing antibodies in vivo using vaccinia virus recombinants for immunization. In addition, the immunodominant neutralizing epitope revealed by the immunization of mice with WRRT may be of significance in vivo and may provide an important basis for genetic immunization in humans. In cases where enzyme-neutralizing antibodies are expected to be effective in preventing microbial infection in humans, this immunization method using the vaccinia virus recombinant could be effectively applied. It is quite interesting to determine whether immunization of humans with WRRT results in the production of antibody to HRSINE. Two human recombinant Fab fragments that are reactive with HIV-1 RT and completely neutralize RT activity have been selected from a synthetic Fab phage display library (Gargano et al., 1996 ). The human Fab fragments, however, recognize a structural fold that is common to the different DNA polymerase and is necessary for their activity (Gargano et al., 1996
). This epitope is apparently closely located to, but different from, HRSINE, since the latter is specific for HIV-1 RT among various DNA polymerases. By immunization with WRRT and thus by expanding human B cell clones that recognize HRSINE, the resultant Fab fragments isolated may be useful for exploring alternative therapeutic strategies based on either gene therapy (Cattaneo & Biocca, 1999
) or recombinant proteins. It should be noted, however, that the immunization protocol employed in this study, namely repeated intravenous immunization, is not practical in humans. Recent success in the development of an expression vector using highly attenuated vaccinia viruses (Moss, 1996
; Sugimoto & Yamanouchi, 1994
) and vectors for plasmid immunization (Ledley, 1995
) encourage us to attempt the establishment of an immunization method to induce enzyme-neutralizing antibodies in humans. It is still unclear why the immunization of mice with the vaccinia virus recombinant is so effective at inducing the clonal expansion of rare B cell clones that produce enzyme-neutralizing antibodies. Further work is required to elucidate the mechanism underlying this efficient clonal expansion.
This unique epitope, named HRSINE, which is functionally significant for RT activity, is an excellent target for the development of new types of HIV-1 RT inhibitors. Moreover, these recombinant antibody fragments may themselves serve as strong and specific inhibitors against HIV-1 replication when expressed within human cells as either intrabodies or intracellular antibodies (Cattaneo & Biocca, 1999 ; Rondon & Marasco, 1997
).
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Acknowledgments |
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Footnotes |
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References |
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Received 15 September 2000;
accepted 15 December 2000.
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