Human neutralizing human immunodeficiency virustype 2-specific Fab molecules generated by phage display

Ewa Björling1,2, Eva von Garrelts3, Andreas Mörner1,2, Mariethe Ehnlund1 and Mats A. A. Persson3

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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
A panel of human immunodeficiency virus type 2 (HIV-2)-neutralizing, recombinant Fab fragments was generated by using the phage display technique. The combinatorial library was derived from an asymptomatic, HIV-2-seropositive individual and constructed on the surface of filamentous phage by using the pComb3 phagemid vector and then screened against native HIV-2 envelope glycoprotein (gp125). Ten of 30 Fab fragments generated displayed strong reactivity in an ELISA and were therefore selected for further study. Six of these possessed neutralizing capacity, with titres varying from 20 to 80 against the homologous HIV-2 strain, and one also had a weak neutralizing capacity against a heterologous HIV-2 isolate, K135. Sequencing of the heavy chain CDR3 regions showed that the gp125-specific Fabs represented individual clones. These reagents may be useful for studies on the conformational structures of the HIV-2 envelope antigens and their immunogenicity, which may help in vaccine design. Furthermore, the cloned Fab genes may be transformed into whole IgG for eukaryotic expression, and as such used for therapeutic and immunoprophylactic studies in HIV-2-infected macaques and, possibly, for human immunoprophylaxis against HIV-2.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
The humoral immune response to human immunodeficiency virus (HIV) is directed mainly against the viral envelope glycoproteins, which contain several targets for neutralizing antibodies. Antibodies responsible for the neutralizing activity against HIV-1 have been shown predominantly to identify discontinuous sites. Of particular importance in this context may be discontinuous structures responsible for CD4 binding in the native gp120 (Steimer et al., 1991 ; Kang et al., 1991b ; McKeating et al., 1992 ; Nakamura et al., 1992 ; Thali et al., 1992 ; Posner et al., 1993 ). Anti-CD4 antibodies seem to be associated with long-term-controlled infection and are lost in individuals with advanced disease (Cavacini et al., 1993 ). Antibodies directed against the CD4-binding region have been shown to block infectivity by inhibiting the gp120–CD4 interaction (reviewed by Moore & Ho, 1995 ).

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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Bone marrow donor.
Bone marrow lymphocytes were obtained from a 50 year old, HIV-2-positive Caucasian male infected in Africa in 1989 and diagnosed by PCR in 1991. At the time of bone marrow donation, he was asymptomatic, with a normal CD4 cell count and a CD4/CD8 ratio of 0·67; he was receiving no medication at this time.

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).

{blacksquare} 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 Tris–HCl, 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.

{blacksquare} 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 ).

{blacksquare} 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 ).

{blacksquare} 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 carbonate–bicarbonate 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 0·01–10 µg/ml and incubated for 1 h at room temperature. After washing, the conjugate AP–goat 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.

{blacksquare} 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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Table 1. Antibody reactivity of human serum 5247 against peptides representing different parts of gp125 and gp36 of HIV-2SBL6669-ISY in a peptide ELISA

 
{blacksquare} ELISA for detection of gp125-reactive antibodies.
ELISAs with GNA-purified gp125 or peptides corresponding to different selected regions in gp125 and gp36 of HIV-2 were performed to investigate the reactivity against the human HIV-2 antibody-positive serum derived from the same patient as the bone marrow sample, and furthermore against the ten selected Fab fragments. Sera from four healthy, HIV-negative Swedish blood donors were used as negative controls. High binding 96-well ELISA plates (Greiner) were coated overnight at room temperature with peptides diluted in 0·01 M carbonate buffer (pH 9·6) to 1 µg per well, or gp125 at 0·25 µg per well, and then blocked with 5% BSA in PBS for 2 h at 37 °C to reduce unspecific binding. Sera and Fabs were diluted 1:100 and 1:5, respectively, in PBS with 20% foetal calf serum, 0·5% BSA and 0·05% Tween 20 and incubated for 1 h at 37 °C. After washing six times, peroxidase-conjugated rabbit Ig to human IgG diluted 1:800 or AP–goat anti-human F(ab')2 (Pierce) diluted 1:500 was added and the plates were incubated for 1 h at 37 °C. The substrate, o-phenylenediamine or p-nitrophenylphosphate solution, respectively, was added and the reaction was stopped with 2·5 M H2SO4 or 1 M NaOH, respectively, after 10 min. Samples that displayed values above the mean A490 or A405 +3 SD of negative human sera were considered positive.

{blacksquare} 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 AP–goat 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.

{blacksquare} 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 HCl–glycine (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.

{blacksquare} 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 virus–Fab 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.

{blacksquare} 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).

{blacksquare} Sequencing of the HIV-2 isolate.
The V3 region of the donor HIV-2 isolate was amplified by RT–PCR 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).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Construction of a human library and selection of antigen binders against HIV-2 gp125
One asymptomatic, HIV-2 antibody-positive individual was chosen for the generation of a combinatorial antibody library. Cloning of Ig fragments into the pComb3 vector resulted in a library of 4x107 members and Fab expression was confirmed by assaying the phage preparation in ELISA. Panning of the library was performed by using a biotinylated gp125 HIV-2SBL6669 protein reacting with the phage in solution, which was shown to be a more efficient screening for antigen binders than coating the antigen to microtitre wells. Antigen and bound phage were coupled to streptavidin-coated paramagnetic beads and the phage were eluted and further propagated in E. coli. Four rounds of panning resulted in a more than 75-fold amplification of eluted phage, suggesting an enrichment of antigen-specific clones. After removal of the gene III fragment, clones were first assayed for Fab production in ELISA. Thirty clones were positive in the assay with antigen in solution, and ten of these were selected for further characterization.

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 8–43% 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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Table 2. Antigenic reactivity of selected Fab clones against gp125 of HIV-2SBL6669

 
Neutralizing capacity of human anti-HIV-2 Fab fragments
Ten clones were selected from the phage display library for their ability to bind gp125 coated onto magnetic beads. Fab supernatants were prepared and then tested for homologous, heterologous and cross-neutralizing ability against HIV-2SBL6669, HIV-2K135 and SIVsm. Neutralization assays were repeated twice with purified clones, with reproducible results. Negative controls included one human HIV-2-non-specific Fab clone and as positive controls we used two anti-HIV-2 sera, one of them homologous to the combinatorial library source. Of ten human anti-HIV-2 clones analysed, two had neutralizing capacity against homologous HIV-2 isolate SBL6669 with a titre of 40 and one Fab, clone 88, had a titre of 80, and also showed a weak neutralizing capacity against a heterologous isolate, HIV-2K135. No cross-neutralization of SIVsm was seen for any of the clones tested (Table 3).


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Table 3. Neutralizing capacity of human Fab fragments directed against gp125 of HIV-2SBL6669

 
Sequencing of heavy chain variable regions of different Fab fragments
By sequencing the complementarity-determining region 3 (CDR3) of the heavy chains, we could conclude that the human Fab fragments isolated represented individual clones (Table 4). At the time of bone marrow drawing, the homologous human serum reacted with several HIV-2 V3 peptides and gp125 and also showed a distinct neutralizing capacity against HIV-2SBL6669, the virus used for panning of the library.


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Table 4. Amino acid sequences of heavy chain CDR3 regions and flanking framework regions FR3 and FR4 of selected HIV-2 gp125-specific Fab fragments

 
Sequence of the donor virus isolate
The nucleic acid sequence of the V3 region of gp125 from the donor isolate was determined. The deduced peptide sequence was CKRPENKTGVQITLRSGLRFHSQGPINSKPKQAWC.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
In this study, we have developed human Fab molecules with neutralizing capacity against HIV-2 by the use of the combinatorial library technique. This method has been used extensively for the preparation of human MAbs against several viruses (reviewed by Parren & Burton, 1997 ). The crucial points before preparation of an immune antibody phage display library are to identify optimal donors and to choose the B-cell source. In this case, it was preferably a person with a controlled HIV-2 infection and a high titre of neutralizing serum antibodies against HIV-2, which is presumed to reflect a vigorous immune response and high levels of specific mRNA for the start of the cloning process. We used B cells from bone marrow from an immune donor with a neutralizing titre of 80 against the heterologous SBL6669 isolate, since our gp125 antigen used for panning was purified from the same isolate. Bone marrow has been shown to be the major depository of antibody-producing cells in humans (Lum et al., 1990 ) and antibodies to many different antigens have been derived from bone marrow libraries constructed from a single donor (Williamson et al., 1993 ).

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.


   Acknowledgments
 
We are indebted to Dr Martin Glennie, Southampton, UK, for the generous gift of rabbit anti-Fab serum. This study was supported by the Tobias Foundation, Physicians against AIDS, the Swedish Society for Medical Research, the Swedish Cancer Society, the Swedish Research Council for Technological Sciences, the Swedish Medical Research Council and the Sven and Dagmar Salén Foundation.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 11 January 1999; accepted 5 April 1999.