Author for correspondence: Hans de Haard. Present address: Department of Functional Biomolecules, Unilever Research Laboratorium Vlaardingen, PO Box 114, 3130 AC Vlaardingen, The Netherlands. Fax +31 10 4605383. e-mail hans-de.haard@unilever.com
Monoclonal antibodies play an important role in the development of diagnostic assays. Instead of using hybridoma technology to isolate human immunodeficiency virus type 1-specific antibodies, a phage-displayed antibody library was generated from a small number (107) of peripheral blood lymphocytes from a seropositive donor. Two families of singlechain antibodies (scFvs) were selected by biopanning with the envelope precursor gp160. ELISA and competition in the BlAcore system revealed that one antibody family recognized a conformation-sensitive epitope within gp120, while the other antibody family was gp41-specific. The latter group had sequence similarity to antibodies recognizing the cluster III epitope of gp41. Binding of scFvs to gp160 could be inhibited with the donor’s serum antibodies, indicating that antibodies with a similar specificity were circulating in the donor’s blood. Competition experiments suggested that the epitope of the anti-gp41 antibodies was recognized by a broad range of patients’ sera: 21 out of 22 sera from North American and all 20 sera from African seropositive patients inhibited binding of scFvs. In contrast, three sera from this panel did not react with the epitope of the anti-gp120 antibodies. These data indicate that, because of the conserved nature of its epitope, the anti-gp41 antibody will be suitable for diagnostic applications.
BarbasC. F., ColletT. A., AmbergW., RobenP., BinleyJ. M., HoekstraD., CababaD., JonesT. M., WilliamsonA., PilkingtonG. R., HaigwoodN. L., CabezasE., SatterhwaitA. C., SanzI., BurtonD. R.1993; Molecular profile of an antibody response to HIV-1 as probed by combinatorial libraries. Journal of Molecular Biology 230:812–823
BaumbergerC., Kinloch-de-LoesS., YerlyS., HirschelB., PerrinL.1993; High levels of circulating RNA in patients with symptomatic HIV-1 infection. AIDS 7: Suppl. 2 S59–S64
BinleyJ. M., KlasseP. J., CaoY., JonesI., MarkowitzM., HoD. D., MooreJ. P.1997; Differential regulation of the antibody responses to Gag and Env proteins of human immunodeficiency virus type 1. Journal of Virology 71:2799–2809
BurtonD. R., BarbasC. F., PerssonM. A. A., KoenigS., ChanockR. M., LernerR. A.1991; A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic individuals. Proceedings of the National Academy of Sciences, USA 88:10134–10137
BuschM. P., AlterH. J.1995; Will human immunodeficiency virus p24 antigen screening increase the safety of the blood supply and, if so, at what cost?. Transfusion 35:536–539
ChargelegueD., ColvinB. T., O’TooleC. M.1993; A 7-year analysis of anti-Gag (p17 and p24) antibodies in HIV-1-seropositive patients with haemophilia : immunoglobulin G titre and avidity are early predictors of clinical course. AIDS 7: Suppl. 2 S87–S90
ChirgwinJ. M., PrzybylaA. E., MacDonaldR. J., RutterW. J.1979; Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299
CoxJ. P. L., TomlinsonI. M., WinterG.1994; A directory of human germ-line VK segments reveals a strong bias in their usage. European Journal of Immunology 24:827–836
CummingS. A., McPheeD. A., MaskillW. J., KempB. E., DohertyR. R., GustI. D.1990; Use of a conserved immunodominant epitope of HIV surface glycoprotein gp41 in the detection of early antibodies. AIDS 4:83–86
de HaardJ. J. W., KazemierB., KoolenM. J. M., NijholtL. J., MeloenR. H., van GemenB., HoogenboomH. R., ArendsJ.-W.1998b; Selection of recombinant, library derived antibody fragments against p24 for application in HIV-1 diagnostics. Clinical and Diagnostic Laboratory Immunology in press
de HaardJ. J. W., KazemierB., van der BentA., OudshoornP., BoenderP., van GemenB., ArendsJ.-W., HoogenboomH. R. M.1998c; Absolute conservation of residue number 6 of immunoglobulin heavy chain variable regions of class IIA is required for correct folding. Protein Engineering in press
DeveyM. E., StewardM. W.1988; The role of antibody affinity in the performance of solid phase assays. In ELISA and Other Solid Phase Immunoassays pp. 135–153KemenyD. M., ChallacombeS. J. Edited by London: John Wiley;
DitzelH. J., BinleyJ. M., MooreJ. P., SodroskiJ., SullivanN., SawyerL. S., HendryR. M., YangW. P., BarbasC. F., BurtonD. R.1995; Neutralizing recombinant human antibodies to a conformational V2- and CD4-binding site-sensitive epitope of HIV-1 gp120 isolated by using an epitope-masking procedure. Journal of Immunology 154:893–906
DitzelH. J., ParrenP. W., BinleyJ. M., SodroskiJ., MooreJ. P., BarbasC. F.IIIBurtonD. R.1997; Mapping the protein surface of human immunodeficiency virus type 1 gp120 using human monoclonal antibodies from phage display libraries. Journal of Molecular Biology 267:684–695
DowerW. J., MillerJ. F., RagsdaleC. W.1988; High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Research 16:6127–6145
EvanG. I., LewisG. K., RamsayG., BishopJ. M.1985; Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Molecular and Cellular Biology 5:3610–3616
FelgenhauerM., KohlJ., RukerF.1990; Nucleotide sequences of the cDNAs encoding the V-regions of H- and L-chains of a human monoclonal antibody specific to HIV-1-gp41. Nucleic Acids Research 18:4927
FischlM. A., RichmanD. D., HansenN., CollierA. C., CareyJ. T., ParaM. F., HardyW. D., DolinR., PowderlyW. G., AllanJ. D., WongB., MeriganT. C., McAuliffeV. J., HyslopN. E., RhameF. S., BalfourH. H., SpectorS. A., VolberdingP., PettinelliC., AndersonJ.1990; The safety and efficacy of zidovudine (AZT) in the treatment of subjects with mildly symptomatic human immunodeficiency virus type 1 (HIV) infection. A double-blind, placebo-controlled trial. Annals of Internal Medicine 112:727–737
ForsterS. M., OsborneL. M., Cheingsong-PopovR., KennyC., BurnellR., JeffriesD. J., PinchingA. J., HarrisJ. R., WeberJ. N.1987; Decline of anti-p24 antibody precedes antigenaemia as correlate of prognosis in HIV-1 infection. AIDS 1:235–240
GnannJ. W.JrMcCormickJ. B., MitchellS., NelsonJ. A., OldstoneM. B.1987; Synthetic peptide immunoassay distinguishes HIV type 1 and HIV type 2 infections. Science 237:1346–1349
GraziosiC., PantaleoG., ButiniL., DemarestJ. F., SaagM. S., ShawG. M., FauciA. S.1993; Kinetics of human immunodeficiency virus type 1 (HIV-1) DNA and RNA synthesis during primary HIV-1 infection. Proceedings of the National Academy of Sciences, USA 90:6405–6409
HoogenboomH. R., GriffithsA. D., JohnsonK. S., ChiswellD. J., HudsonP., WinterG.1991; Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Research 19:4133–4137
HortonR. M., HuntH. D., HoS. N., PullenJ. K., PeaseL. R.1989; Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68
JanvierB., BaillouA., ArchinardP., MounierM., MandrandB., GoudeauA., BarinF.1991; Immune response to a major epitope of p24 during infection with human immunodeficiency virus type 1 and implications for diagnosis and prognosis. Journal of Clinical Microbiology 29:488–492
KabatE. A., WuT. T., PerryH. M., GottesmanK. S., FoellerC.1991Sequences of Proteins of Immunological Interest, 5th edn. Bethesda, MD: US Department of Health and Human Services, Public Health Service, National Institutes of Health;
KalyanaramanV. S., PalR., GalloR. C., SarngadharanM. G.1988; A unique human immunodeficiency virus culture secreting soluble gp160. AIDS Research and Human Retroviruses 4:319–329
KalyanaramanV. S., RodriguezV., VeroneseF., RahmanR., LussoP., DeVicoA. L., CopelandT., OroszlanS., GalloR. C., SarngadharanM. G.1990; Characterization of the secreted, native gp120 and gp160 of the human immunodeficiency virus type 1. AIDS Research and Human Retroviruses 6:371–380
LewA. M.1984; The effect of epitope density and antibody affinity on the ELISA as analysed by monoclonal antibodies. Journal of Immunological Methods 72:171–176
LouwagieJ., JanssensW., MascolaJ., HeyndrickxL., HegerichP., van der GroenG., McCutchanF. E., BurkeD. S.1995; Genetic diversity of the envelope glycoprotein from human immunodeficiency virus type 1 isolates of African origin. Journal of Virology 69:263–271
MarksJ. D., HoogenboomH. R., BonnertT. P., McCaffertyJ., GriffithsA. D., WinterG.1991; By-passing immunization: human antibodies from V-gene libraries displayed on phage. Journal of Molecular Biology 222:581–597
MullinaxR. L., GrossE. A., AmbergJ. R., HayB. N., HogrefeH. H., KubitzM. M., GreenerA., AltingM. M., ArdourelD., ShortJ. M., SorgeJ. A., ShopesB.1990; Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpression library. Proceedings of the National Academy of Sciences, USA 87:8095–8099
MusterT., SteindlF., PurtscherM., TrkolaA., KlimaA., HimmlerG., RukerF., KatingerH.1993; A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. Journal of Virology 67:6642–6647
ParrenP. W., BurtonD. R.1997; Antibodies against HIV-1 from phage display libraries : mapping of an immune response and progress towards antiviral immunotherapy. Chemical Immunology 65:18–56
PerssonM. A., CaothienR. H., BurtonD. R.1991; Generation of diverse high-affinity human monoclonal antibodies by repertoire cloning. Proceedings of the National Academy of Sciences, USA 88:2432–2436
SchiblerU., MarcuK. B., PerryR. P.1978; The synthesis and processing of the messenger RNAs specifying heavy and light chain immunoglobulins in MPC-11 cells. Cell 15:1495–1509
SchmidtG., AmiraianK., FreyH., WethersJ., StevensR. W., BernsD. S.1989; Monitoring human immunodeficiency virus type 1-infected patients by ratio of antibodies to gp41 and p24. Journal of Clinical Microbiology 27:843–848
SkerraA., PluckthunA.1991; Secretion and in vivo folding of the Fab fragment of the antibody McPC603 in Escherichia coli: influence of disulphides and cis-prolines. Protein Engineering 4:971–979
SpectorS. A., KennedyC., McCutchanJ. A., BozzetteS. A., StraubeR. G., ConnorJ. D., RichmanD. D.1989; The antiviral effect of zidovudine and ribavirin in clinical trials and the use of p24 antigen levels as a virologic marker. Journal of Infectious Diseases 159:822–828
StrathdeeS. A., FrankJ. W., McLaughlinJ., LeblancM., MajorC., O’ShaughnessyM. V., ReadS. E.1995; Quantitative measures of human immunodeficiency virus-specific antibodies predict progression to AIDS. Journal of Infectious Diseases 172:1375–1379
TolmanR. L., BednarekM. A., JohnsonB. A., LeanzaW. J., MarburgS., UnderwoodD. J., EminiE. A., ConleyA. J.1993; Cyclic V3 loop-related HIV-1 conjugate vaccines. Synthesis, conformation and immunological properties. International Journal of Peptide and Protein Research 41:455–466
TomlinsonI. M., WalterG., MarksJ. D., LlewelynM. B., WinterG.1992; The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops. Journal of Molecular Biology 227:776–798
VolberdingP. A., LagakosS. W., KochM. A., PettinelliC., MyersM. W., BoothD. K., BalfourH. H. J., ReichmanR. C., BartlettJ. A., HirschM. S., MurphyR. L., HardyW. D., SoeiroR., FischlM. A., BartlettJ. G., MeriganT. C., HyslopN. E., RichmanD. D., ValentineF. T.1990; Zidovudine in asymptomatic human immunodeficiency virus infection. A controlled trial in persons with fewer than 500 CD4-positive cells per cubic millimeter. New England Journal of Medicine 322:941–949
Von SydowM., GainesH., SonnerborgA., ForsgrenM., PehrsonP. O., StrannegardO.1988; Antigen detection in primary HIV infection. British Medical Journal Clinical Research 296:238–240
WatkinsB. A., BugeS., AldrichK., DavisA. E., RobinsonJ., ReitzM. S.JrRobert-GuroffM.1996; Resistance of human immunodeficiency virus type 1 to neutralization by natural antisera occurs through single amino acid substitutions that cause changes in antibody binding at multiple sites. Journal of Virology 70:8431–8437