Microbiology and Tumorbiology Center (MTC), Karolinska Institute, S-171 77 Stockholm, Sweden1
Department of Immunology, Microbiology, Pathology and Infectious Diseases (IMPI), Karolinska Institute, Huddinge, Sweden2
Department of Medicine, Karolinska Institute, Karolinska Hospital (L8:01), S-171 76 Stockholm, Sweden3
Author for correspondence: Ewa Björling (at the Microbiology and Tumorbiology Center). Fax +46 8 33 07 44. e-mail ewa.bjorling{at}mtc.ki.se
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Abstract |
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Introduction |
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HIV-2, the second lentivirus that causes immunodeficiency in humans, was first isolated from a West African patient (Clavel et al., 1986 ; Kanki et al., 1987
; Albert et al., 1987
). HIV-2 infection has today been documented in Africa, Europe, the Americas and Asia, but is still largely confined to West Africa and Portugal. HIV-2 infections, like HIV-1 infections, result in the production of neutralizing antibodies predominantly directed against regions in the envelope glycoprotein (Weiss et al., 1985
; Ranki et al., 1987
) and there is also evidence in vitro of cross-neutralizing antibodies (Weiss et al., 1988
; Böttiger et al., 1989
, 1990
).
Conflicting results have been presented by several groups concerning the ability of peptides or recombinant proteins representing the HIV-2 V3 region to elicit neutralizing antibodies (reviewed by Kent & Björling, 1997 ). We have shown previously that the third variable region (V3) in the surface glycoprotein of HIV-2 contains two immunodominant overlapping sites important for neutralizing antibodies that may alternatively act together to form a discontinuous epitope. Furthermore, peptides representing the V3 region can block the neutralizing capacity of human anti-HIV-2 sera (Björling et al., 1991
, 1994
). These findings were supported by Matsushita et al. (1995)
, who developed a MAb (B2C) directed against the HIV-2 V3 region. This MAb was capable of neutralizing both cell-free and cell-associated virus infections in an isolate-specific fashion. In another report, McKnight and co-workers described the production of rat MAbs directed against both V3 and conformational epitopes on the HIV-2 envelope glycoprotein by immunization with recombinant baculovirus-derived gp105. These MAbs have also been shown to harbour some neutralizing capacity against HIV-2 and simian immunodeficiency virus (SIV) (McKnight et al., 1996
). One human conformational MAb with neutralizing capacity against HIV-2 has also been developed (Kent et al., 1993
).
In this presentation, we constructed a human antibody library by the phage display technique from which we generated recombinant human Fab fragments that reacted with gp125 of HIV-2SBL6669. Characterization of the isolated Fab fragments showed that several had strong neutralizing capacity against the homologous isolate HIV-2SBL6669-ISY, and sequencing of the Fabs showed that they were clonally distinct from each other.
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Methods |
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Five millilitres of bone marrow was collected by aspiration. RNA was recovered from the cells by using a total RNA isolation system (Stratagene) based on acid phenol extraction (Chomczynski & Sacchi, 1987 ) and first-strand cDNA synthesis was performed by oligo(dT) priming of 5 µg RNA (cDNA synthesis kit, Pharmacia).
PCR protocol.
A standard protocol was used for amplification of immunoglobulin (Ig) cDNA. PCR buffer, containing 1·5 mM MgCl2, 0·05 M KCl, 0·01 M TrisHCl, pH 9·1, and 0·1% Tween 20, was mixed with dNTP (200 µM of each nucleotide), 5 U Taq polymerase (Perkin-Elmer Cetus) and 400 nM of the appropriate primers in a total volume of 100 µl. The reaction mixtures were heated to 94 °C for 5 min and then subjected to 35 rounds of amplification (Hybaid thermal cycler) of 94 °C for 1 min, 52 °C for 30 s and 72 °C for 3 min, followed by a final incubation at 72 °C for 10 min.
Library construction.
Human heavy (Fd) and light chain cDNAs were PCR-amplified by using 5'-biotinylated primers designed for the amplification of human Ig (Kang et al., 1991a ). After restriction enzyme digestion of heavy and light chain DNAs (XhoI/SpeI and SacI/XbaI, respectively), removal of incomplete fragments was achieved by adsorption to streptavidin-coated paramagnetic beads (Dynal). Heavy and light chains were cloned into the pComb3 vector as described previously (Barbas et al., 1991
; Burton et al., 1991
). After transformation by electroporation of XL-1 Blue cells (Barbas & Lerner, 1991
), 3 ml SOC medium (Sambrook et al., 1989
) was added and the culture was incubated for 1 h. The culture was then transferred to 10 ml SB medium (super broth; Burton et al., 1991
) containing ampicillin (20 µg/ml), tetracycline (10 µg/ml) and 1% glucose and incubated for 1 h. Ninety millilitres of SB containing ampicillin (50 µg/ml) and tetracycline (10 µg/ml) was added and the culture was incubated for 2 h at 37 °C in a shaker. All incubations were done at 37 °C on a rotating platform. Helper phage (2x1011 p.f.u. VCS-M13, Stratagene) and 2 mM IPTG were added and the culture was grown overnight at 30 °C. The resulting phage were recovered as described previously (Burton et al., 1991
).
Antibody selection by panning of the library.
For the selection procedure for antigen binders, we used a modified panning protocol described previously by Samuelsson et al. (1995) . Lectin-purified (Galanthus nivalis agglutinin; GNA) HIV-2 envelope glycoprotein, gp125, was biotinylated by using NHS-LC-biotin reagent (Pierce). To eliminate unspecific binders, 100 µl phage suspension was preadsorbed to streptavidin-coated paramagnetic beads (Dynal) that had been blocked for 1 h in 3% BSA and 3 µg/ml streptavidin (Pierce) in PBS. The unadsorbed phage were incubated with 1 µg biotinylated antigen and 1% BSA in PBS in a total volume of 200 µl for 2 h at room temperature. Biotinylated antigen and bound phage were coupled to 50 µl milk-blocked streptavidin beads by incubating for 15 min at 37 °C and resuspending twice during this time. Unbound phage were discarded and the beads were washed once in water. The beads were further washed ten times in PBS with 0·05% Tween 20 (PBS-T) and once in water. Bound phage were eluted with 100 µl elution buffer (0·1 M HCl adjusted to pH 2·2 with glycine, with 0·1% BSA) for 10 min. The eluate removed from the beads was neutralized with 14 µl 2 M Tris base and used for infection of 3 ml fresh XL-1 Blue cells (OD600 of 1) for 15 min at room temperature. The infected cells were transferred to 10 ml SB containing 20 µg/ml ampicillin and 10 µg/ml tetracycline. Samples (5 and 0·01 µl) were removed for plating to calculate the number of eluted phage. The culture was grown for 1 h at 37 °C and then added to 100 ml SB with 50 µl/ml ampicillin and 10 µg/ml tetracycline. After shaking at 37 °C for 1 h, helper phage was added as described above and recovered after overnight culture at 30 °C. Phage were prepared and used for repeated panning. After completion of the panning procedure, the removal of the gene III fragment, propagation of single clones and induction of Fab production were performed as described previously (Burton et al., 1991
; Collet et al., 1992
).
ELISA for Fab concentration.
An ELISA for the determination of Fab concentration has been detailed elsewhere (Samuelsson et al., 1995 ). Briefly, goat anti-human F(ab')2 (Pierce) or goat anti-human Fd (The Binding Site) was diluted 1:1000 in 0·1 M carbonatebicarbonate buffer, pH 9·6, coated onto microtitre plates (100 µl per well) and incubated overnight at 4 °C. The coating solution was discarded and the plates were blocked with 5% milk powder in PBS. The blocking solution was removed and samples, at appropriate dilutions in PBS-T, or purified polyclonal human Fab (Nordic) were added at 10-fold dilutions of 0·0110 µg/ml and incubated for 1 h at room temperature. After washing, the conjugate APgoat anti-human F(ab')2 (Pierce), diluted 1:500, was added after incubation for 1 h at 37 °C. After five washes, the substrate solution, p-nitrophenylphosphate (Sigma) in 0·1 M diethanolamine, pH 9·8, was added. Absorbance was measured at 405 nm.
Peptides.
Thirteen peptides (Table 1), corresponding to selected previously described, overlapping antigenic sites for human sera in the envelope glycoprotein of HIV-2 (Norrby et al., 1991
), were synthesized by the solid-phase multiple peptide method using t-Boc chemistry. The peptide synthesis was done as described previously (Björling et al., 1991
).
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Competitive enzyme immunosorbent assay (EIA) for gp125 specificity.
The specificity and relative affinity of the Fab molecules were estimated by using an inhibition EIA utilizing paramagnetic beads as described previously for the phage selection procedure (outlined above) (Rath et al., 1988 ). Fab molecules were incubated with biotinylated gp125; in parallel vials, 25 nM non-biotinylated gp125 was also included. After 2 h at room temperature, the beads were washed in PBS-T and incubated for 1 h at room temperature with APgoat anti-human F(ab')2 (Pierce) diluted 1:500. After five washes, substrate solution, p-nitrophenylphosphate (Sigma) in 0·1 M diethanolamine, pH 9·8, was added. Absorbance was measured at 405 nm.
Purification of Fab fragments.
An affinity column was generated by linking 80 mg total protein from the Ig preparation (IgG fraction) of serum derived from rabbits immunized with polyclonal human Fab fragments (kindly donated by M. Glennie, University of Southampton, UK) to 5 ml Affi-gel 10 according to the manufacturer's instructions (Bio-Rad). Crude Fab preparations of bacteria were diluted in PBS, filtered (0·45 µm) and applied to the column. The column was washed with ten column volumes of PBS and then with five volumes of PBS containing 0·5 M NaCl. Bound Fab molecules were eluted with 0·1 M HClglycine (pH 2·2). Fractions were collected and neutralized immediately with 2 M Tris base. Appropriate Fab-containing fractions were pooled and dialysed overnight. The preparations were concentrated by using Centricon 30 centrifugal microconcentrators (Amicon) centrifuged at 5000 g for 1 h. The resulting Fab preparations were analysed for Fab concentration, antigen reactivity and neutralizing activity against HIV-2 and SIV isolates.
Neutralization assay.
The diluted tissue culture supernatant of HIV-2SBL6669-, HIV-2K135- or SIVsm-infected peripheral blood mononuclear cells (PBMC) (50 TCID50, 100 µl) was incubated for 2 h at 37 °C with 2-fold serial dilutions of Fab fragments starting at a dilution of 1:20. PBMC (1x105 in 50 µl) were added to the virusFab fragment reaction mixture and incubated overnight. All dilutions were performed in RPMI 1640 medium (Gibco) supplemented with 10% foetal calf serum, 3 mM glutamine, IL-2 and antibiotics. This medium mixture was also used for cell culture during the experiment. Medium changes were performed on days 1 and 4. Seven days after infection, supernatants were collected and analysed by a modified HIV-2 antigen-capture ELISA (Thorstensson et al., 1991 ). The neutralization titre was defined as the last dilution step that showed a reduction in A490 of 80% or more in the culture supernatant compared with the respective negative control Fab or HIV antibody-negative serum. Titres above 20 were considered to represent positive neutralization and assays were repeated at least on two occasions.
Sequencing of Fab fragments.
Plasmid DNA from selected clones was isolated and the Ig DNA-containing part of the plasmids was amplified by PCR with primers that hybridize to the T3 and T7 regions. The T3 primer was biotinylated at the 5' end. PCR amplification was performed as described above and single-stranded DNA was generated according to Hultman et al. (1989) . The single-stranded DNA was used as template for sequencing reactions with primers hybridizing some 90 bp 3' of the junction between the variable and constant regions (SEQGb, 5' GTCGTTGACCAGGCAGCCCAG) for gamma heavy chains; analysis was performed with an ALF automated sequencer (Pharmacia Biotech).
Sequencing of the HIV-2 isolate.
The V3 region of the donor HIV-2 isolate was amplified by RTPCR from the bone marrow lymphocyte RNA obtained from the Fab library donor, using the same reverse transcriptase reagents as when generating the Fab library (see above). The PCR used was a nested PCR, utilizing primers JA163 and JA166 as outer primers and JA164 and JA165B in the second PCR as inner primers (Albert et al., 1996 ). The sequence of the resulting PCR product was determined by using either of the inner primers in cycle sequencing reactions (Perkin-Elmer). The resulting products were analysed on an ABI 377 automatic sequencer (Perkin-Elmer).
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Results |
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At the time of blood and bone marrow drawing, the patient was clinically healthy with a normal CD4 cell count and showed distinctive serologic antibody reactivity against gp125 and several peptides corresponding to selected antigenic regions in gp125 of HIV-2SBL6669.
Peptide ELISA
Serum (5247) from the HIV-2-positive bone marrow donor was tested against thirteen peptides corresponding to selected regions in gp125 and gp36 of HIV-2. Nine peptides of thirteen reacted positively in the ELISA, with absorbances ranging from 0·50 to 2·86 after subtraction of values obtained with HIV-negative sera. The most prominent antigenic activity was seen against peptides mimicking the V3 region of gp125 (A4-34, A4-37, A5-3 and A5-8; Table 1). The same peptides were also tested against all ten Fab fragments, none of them showing any antigenic reactivity.
Specificity of anti-gp125 Fab clones
The specificity of the selected clones was ascertained by using a competitive EIA, where non-biotinylated gp125 competed with the corresponding biotinylated form for interaction with the Fab molecules. Of 30 clones tested, 10 were clearly specific, i.e. their binding to gp125 was inhibited by 843% in the presence of 25 nM free antigen (Table 2). In addition, this indicated that the affinities for the tested clones were less than 107 mol-1.
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Discussion |
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To begin with, we selected thirty Fabs directed against gp125 of HIV-2SBL6669. Ten of these Fabs showed strong reactivity in ELISA and were therefore selected for further study. Six of these had neutralizing capacity, with titres varying from 20 to 80 against the homologous HIV-2 strain, and one Fab had also neutralizing capacity against a heterologous HIV-2 isolate. These Fab clones were then purified, and sequencing of heavy chain CDR3 regions revealed that all gp125-specific Fabs represented individual clones.
The antibodies did not react with the linear peptides tested, though such specificities was present in serum of the donor. Instead, the Fab clones isolated are likely to react with conformational epitopes. In addition, their neutralizing capacities indicate that they may be reactive towards the envelope protein conformation on the virions, which would be beneficial if they are to be used for passive immunotherapy. If this assumption is correct, they may also be used for assessment of the structure of envelope proteins, e.g. if produced for vaccinations. For passive immunization, it may be beneficial to express them as whole IgG, which has a longer half-life in the organism. In addition, we have previously noted that eukaryotic expression of some human Fab clones isolated by phage display may enhance their efficacy, probably due to imperfect folding of certain Fab molecules in bacteria (Samuelsson et al., 1996 ). Thus, an improvement may be expected if the clones are expressed in eukaryotic cells. Moreover, it will be of interest to investigate whether the clones isolated bind to the same or different epitopes on the envelope glycoprotein, as combinations of antibodies to different epitopes may be preferred in passive immunization trials. In addition, increased affinity has been shown to increase the neutralizing potential of human anti-HIV-1 antibodies (Barbas et al., 1994
); judging from the data from our competitive assay (Table 2
), the strongest-binding Fab clone may have an approximate affinity of 107 mol-1, although this was tested with purified gp125. Still, this clone did not show the greatest neutralizing capacity, and thus affinity to the envelope protein as it is presented on the virion may be more relevant.
The sequence of the V3 region of the donor isolate did not show a close relationship to that of the HIV-2SBL6669 strain used for selection and neutralization tests; 8 of 35 residues were different. Still, six of ten Fab clones neutralized the test strain to various degrees. Possibly, our selection method, which should favour cross-reacting clones, did bias our selection of clones in favour of those able to neutralize HIV-2SBL6669, an isolate unrelated to that of the donor. Indeed, that such clones were present in the donor was indicated by the considerable serum reactivity to linear peptides representing the V3 region of isolate HIV-2SBL6669 (Table 1).
Taken together, we have cloned human MAbs reactive to HIV-2 that neutralize the virus in vitro. Further investigations are needed in order to establish whether these antiviral antibodies may be used successfully for prophylactic or therapeutic studies of HIV-2 infection in macaque and man.
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Acknowledgments |
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References |
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Received 11 January 1999;
accepted 5 April 1999.