FEMS Microbiologyin~nunology64 (1990) 103-110 Publishedby

103

FEMSIM00108

Characterization of monoclonal antibodies to human immunodeficiency virus type I gp41 by HIV-1 polypeptides expressed in Escherichia coli Clara Larcher 1, Michael BriSker 2, Hartwig P. Huemer 1, Brigitte S~lder 1, Thomas F. Schulz 3, Johanna M. Hofbauer !, Helmut Wachter 4 and Manfred P. Dierich 1.4 t

lnstitut fib" Hygiens; University of lnnsbruck, Austria; z Behring Werke AG, Marbur~ F.K G.; ~ The Institute of Cancer Research, Chester Beatty Laboratories, London, U.IC; and 4 Ludwig-Boltzmann-lnstitut f ~ AlDS-Farschung, lnnsbruck, Austria

Received2 March1990 Accepted29 March1990 Key words: HIV-1; gp41; gpl20; Envelope protein; HIV-1 polypeptides expressed in E. coli; Monoclonal antibody 1. SUMMARY Two monoclonal antibodies (MAbs) were produced in Balb/c mice by immunization with recombinant 8p41 derived from expression of ~BH10 eDNA of the human immunodeficiency virus-1 (HIV-1) in the prokaryotic expression vector pEX-41 [1,2]. Characterization of the epitopes recognized by these MAbs was done with HIV-1 envelope (env) fusion proteins expressed in Escherichia coii encoding ten distinct segments of the env proteins [3]. In comparison, another mouse MAb, M25 [4], a human MAb directed against gp41, which was produced by the xeno hybridoma line 3D6 [5,6] and a pool of human patient sera containing antibodies to HIV-1 were tested. We were able to demonstrate that the epitopes recognized by our MAbs are located between arg732 and ser759 [7] of the HIV-1 env glycoprotein 8p160 of HTLV-III strain B. M25 reacted with

Correspondencew: C. Latchet, Institute for Hysiene,University of Innsbruck,Ffitz-Pregl-Str.3.A-6020Innsbmck,Austria.

epitopes between ser647 and pro731, which includes the hydrophobic transmembrane region of gp41 [4]. The human MAb against gp41, 3D6 [5,6] reacts with cpitopes between ile474 and trp646, a polypeptide stretch consisting of gpl20 and gp41 specific amino acids. The human serum pool, positive for HIV-1 antibodies, reacted predominantly with antigenic determinants located between ile474 and 1eu863. The recombinant env fusion proteins were initially produced to test the immunoreactivity with patient sera and to characterize epitopes which are relevant for immunocliagnostic purposes [3]. In this study, we showed that the set of recombinant env proteins is also a simple and accurate tool for the characterization of MAbs directed to the HIV envelope proteins. 2. INTRODUCTION Glycoprotein 41 (gp41) represents the transmembrane portion of the envelope glycoprotein of the human immunodeficiency virus-1 (HIV-1) the etiologic agent of the acquired immune deficiency

0920-8534/90/$03.50 © 1990 Federationof EuropeanMicrobiologicalSq3ciefies

104 syndrome (AIDS) [8,9]. Investigations on gp41 are valuable for several reasons: Antibodies to gp41 are in most cases the first to be detected in HIV infection and in contrast to antibodies to the core protein 1)24, antibody titers do not decrease in sera of full-blown AIDS patients [10-13]. Gp41 seems to be of crucial functional importance. First, it seems to be essential for the entry of HIV-1 into the host cell: after binding of gpl20 to CD4 [14] the fusion process is possible mediated by the transmembrane protein, because the fusogenic property of HIV-1 was found to be abolished after mutations in the 3' end of env (the C terminus of gp41) [15,16]. Second, epitopes located on gp41 probably account for a broadening of the neutralizing antibody response, which has been observed in sera of HIV-1 infected chimpanzees and humans as HIV infection progresses [17-21]. Third, immunosuppressive effects of transmembrane proteins (P15E) were described in vitro in other retroviruses, such as feline leukemia virus or murine leukemia virus [22,23]. These retroviral proteins bear amino acid sequence homologies with the gp41 of HIV-1. Other investigators tested the presence of antibodies to synthetic peptides representing the putatively immunosuppressive part of the 8p41 in HIV-positive persons. They detected a strong positive correlation between health in these persons and prevalence of antibodies against one of the synthetic peptides HIV-env 583-599 (pHIVIS) [24]. MAbs for in vitro and in vivo studies of 81>41 were produced by immunization of Balb/c mice with recombinant !)41 derived from ?~-BH10 cDNA in the prokaryotic expression vector pEX41 [1,2]. Characterization of the MAbs with conventional methods, such as enzyme-linked immunosorbent assay, immunofluorescence assay of HIV-l-infected H9 Cells, commercially available Western Blots (WB) and radioimmunoprecipitation revealed the specificity of these MAbs but did not permit an exact localization of the epitopes recognized by the MAbs [25]. Therefore, we tested the immunoreacfivity of the :'~[A~ with defined segments of envelope glycoproteins of the HIV-1 which were expressed in E. coil [3]. In comparison we tested another mouse MAb against 8p41 produced by diMarzo Veronese [4] and a human

MAb against gp41 [6]. In this study we present a simple and effective method to localize the epitopes of MAbs directed against the envelope of HIV-1 by means of recombinant HIV-envelope proteins.

3. MATERIALS AND METHODS

3.1. Production of hybridomas Balb/c mice (6 weeks of age) were immunized with a fragment of the HIV-1 env (kindly provided by R.C. Gallo) expressed in E. coli: the expression plasmid pEX-41 was constructed as previously described [1,2]. Briefly, a 1.4 kb Bglll fragment (nucleotides 7198-8627 [7]) that codes for the entire 8p41 and an additional 45 amino acids derived from gpl20, was ligated into the BamHl site of pEX2, a bacterial expression vector [26] (kindly provided by Dr. K.K. Stanley), which contains the PR promoter of bacteriophage ?~. The recombinant gp41 used for immunization was purified by a three-step method including iysis of bacteria, fractionated detergent extraction with Triton-X-114 (Scrva) and gel fdtration. The first intraperitoneal inoculation of mice was done with purified gp41 fusion protein (100/tl corresponding to approximately 5 ~rg protein) emulsified in complete Freund's adjuvant. After 3 weeks mice were boosted with the same concentration of antigen emulsified in incomplete adjuvant subcutaneously. The following 5 booster doses were given 10 days apart, alternatively intraperitoneally and subcutaneously. Three days after the final inoculation mice spleen cells were fused with the mouse myeloma line GM5426A. Cell fusion, culturing and cloning of the hybridomas was performed following standard methodology [28]. 3.2. Characterization of hybridomas 3.2.1. Screening assays Screening of supernatants from growing hybridomas for antibodies to gp41 was done by enzyme-linked immunosorbent assay (ELISA, Vironostika anti-HTLV I l l Microelisa System, Organon Teknika) and indirect immunofluorescence. Immunofluorescence was performed with acetone-fixed HIV-1 (HTLV

105

I I I / B ) infected H9 cells and uninfected H9 cells (kindly donated by Dr. Gih'tler, Munich) which were grown in RPIVII-1640 medium containing I 0 ~ fetal calf serum, 2 m M glutamine, I00 U / m l penicillin and 1 0 0 / t g / m l streptomycin. Cells were incubated with hybridoma supernatants for 30 min at room temperature (RT) in a humid atmosphere, then washed with phosphate buffered saline (PBS) and incubated for a further 30 min with fluoresce~n-conjugated rabbit immunoglobufins (lgG) directed to mouse IgG (Tago, Medac) in a dilution of 1 : 20 under the same conditions. 3.2.2. Immunoblot technique SDS-polyacrylamide electrophoresis and blotting techniques (WB) were performed as previously described [29,30] with the following types of antigens: (1) WB of complete HIV-1 preparations purchased from DuPont and Biorad; (2) WB prepared with recombinant entire 8p41 (pEX-41 derived fusion protein); (3) WB prepared with recombinant HIV1 env polypepfides, expressing distinct portions of the ear gene. Defined c D N A sequences encoding segments of the env glycoproteins of HIV-1 were expressed in £. coil as previously described [3]. Briefly, distinct portions of the c D N A coding for the env gene of HIV-1 isolate BH10 [7] were fused in-frame to a truncated lacZ gene coding for the first aminotennina1375 amino acids of/Lgalactosidase [31]. The numbering of the amino acids of the env proteins corresponds to the open reading frame for the env gene of HIV-1 isolate clone BH10 [7], starting from residue lysl to !eu863. The predicted translational start codon of this open r~ading frame is Methionine at position 8 (Fig. 1). The env fusion proteins were expressed by induction of the lac promoter which was achieved by growing the bacteria for 3 - 5 h in 1 m M isopropyl fl-D-thiof,alactoside (IPTG, Sigma). These f u s i o n proteins have previously been found to be highly immunoreactive with patient sera [3]. The recombinant plasmid pMB2440 codes for nearly the total 8p160, from va149 to 1eu863, just missing the sisnal sequence. Plasmid pMB2440 H A differs from plasmid pMB2440 only by the internal deletion of the hydrophobic amino acid sequence of the transmembrane region of 81>41, ser647-pro731. Plasmids pMB254 (val~-arg2s0), pMB256 (va149-

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Fig. 1. (A) Schematic diagram of the env gene of HIV-I, indicating the restriction enzymes used for cloning of the distinct env fragments (upper part). The arrow marks the position of the proteolytic cleavagesite of the precursor prorein gp160 into gpl20 and gp41. The opea bars (lower part) represent the individual parts of the env protein gp160, which were expressed as flga/l-375:eav fusion proteins by the indicauxl ~

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fusion protein encoded by pMB 2440 HA indicates a deletion of a polypeptide stretch encoded by the Hindlll/Aval DNA fragment. (B) Reaction of MAIn 1Cll, 2C7, M25, 31)6 and a pool of human sera containing antibodies against HIV-1 in Western blot ~ with ear fusion proteins encoded by the plasmids of the pMB

106 gly4~3) and pMB580 (ser2st-gly473) code for different parts of gpl20, pMB252 (serTs9-1ens63) for the carboxyterminal of 8t>41, the other plasmids, pMB1790 (va149-trpe4e), pMB242 (ile474-1eus63), pMB261 (ile474-serTsg)and pMB255 (ile474-trp6~s) code for segments of gpl60 including the junction between gp120 and 81>41. After washing the bacteria with PBS they were lysed in PBS containing 1 mg/ml lysozyme (from egg white, ca. 25000 units/mg cryst, Serva), 1~ Triton X-100 (Serva) and 10 pg/ml DNAsel (from bovine pancreas, grade II, Boehringer Mannheim). The proteins from the lysates were separated in 9.5~ SDS-PAGE under reducing conditions and were transferred to nitrocellulose sheets. Supernatants of MAbs 1 C l l arid 2C7 were tested in comparison to mouse MAb M25 (kindly provided by Dr. R.C. Gallo, NIH, Bethesda, MD) [4] and a human MAb directed against gp41 produced by the xeno hybridoma line 3D6. A pool of three human sera containing antibodies against HIV-1 (determined by ELISA and WB assays) and normal human sera were used as controls. Cell culture supernatants of MAbs producing hybridomas were used undiluted, ascitic fluid in a dilution of I : 100, MAb M25 1 : 50, MAb 3D6 1 : 50 and the human antiserum pool 1 : 100.

4. RESULTS

4.1. Characterization of hybridomas 4.1.1. Screening assays The supernatants of hyo bridomas which were found to be positive in ELISAs were further tested by indirect immunofluorescence on acetone-fixed cells: positive clones showed cytoplasmatic immunofluorescence of HIV-1 infected H9 cells. Uninfected H9 cells were not stained. Onunfixed cells membrane fluorescence could not be detected. Two stable hybfidomas, 1Cll and 2C7, of IgG2a subtype were obtained. 4.1.2. Immunoblot assays (1) Reaction with HIV-1 proteins in immunoblots prepared with complete HIV-I: the MAbs 1 C l l and 2C7 stained the bands corresponding to 8P41, 81)160 and a protein of 120 kDa of HIV-1 on WB strips (for refs. see reL 25). The fact that a MAb directed

against 81)41 reacts with bands that develop at gpl60 and gpl20 was already described by ZollaPazner and coworkers [32], who identified the gpl60 and 120 material in commercial WB to be a multimeric form of 8p41. (2) Reaction with the recombinant pEX-41 material used for immunization: both MAbs revealed two proteins with apparent molecular weight of 160 and 41 kDa (for refs. see ref. 25). With these WB it was not possible to determine precisely whether the MAbs were directed against the gp41 transmembrane part of HIV-1 or if they reacted also with the carboxyterminal 45 amino acids of gpl20 which was part of the fusion protein used for immunization. Furthermore, the MAbs precipitated the gpl60 and also a protein of 120 kDa from [35S]methionine-labelled HIV-l-infected H9 cells in a radioimmunoprecipitation assay (RIPA). The possibility that this 120 kDa protein is a multimeric form of 8t>41, as discussed above, cannot be ruled out. A precipitation line corresponding to 1341 could not be visualized by RIPA, probably because of the 18(3 banding at a similar MW position [25]. (3) Reaction of MAbs 1 C l l and 2C7 in comparison to mouse MAb M25 and a human MAb directed to 81>41 with the recombinant env fusion proteins encoded by the plasmids of the pMB series: MAb 1 C l l (identical reaction pattern of 2C7 not shown) reacted with the recombinant env polypeptides encoded by the plasmids pMB2440 (lane 1), pMB2440 HA (lane 2), pMB261 (lane 9) and pMB242 (lane 5) but neither with plasmids pMB255 (lane 8) nor pMB252 (lane 7) (Fig. 2A). This indicates that the epitopes recognized by our two MAbs are located between arg732 and ser759 of the HIV-1 env protein gpl60. MAb M25 reacted with fusion proteins directed by plasmids pMB2440 (lane 1), pMB261 (lane 9) and pMB242 (lane 5), (Fig. 2B) indicating a binding epitope between set647 and pro731, which corresponds to the findinss of Veronese et al. [4]. The human MAb 31)6 reacted with the hybrid proteins encoded by plasmids pMB2440 (lane 1), pMB2440 HA (lane 2), pMB242 (lane 5), pMB261 (lane 9) and pMB255 (lane 8) (fig. 2C) and therefore recognizes an epitope within the polypeptide stretch between ile474 and trp646. The human serum

107 pool, positive for HIV-1 antibodies reacted with the recombinant env proteins encoded by the plasmids pMB2440 (lane 1), pMB2440 HA (lane 2), pMB1790 (lane 6), pMB242 (lane 5), pMB261 (lane 9), pMB255 (lane 8) and pMB252 (lane 7) (Fig. 3). An overview of the reaction pattern of the three MAbs, the human MAb and the human serum pool is given in Fig. lB.

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5. DISCUSSION Reaction of the MAbs 1Cll and 2C7 with pMB2440, pMB2440 HA, pMB242 and pMB261 derived fusion proteins and lack of reaction with plasmids pMB255 and pMB252 (Fig. 2A) indicate that they are directed against epitopes between arg732 and ser759. Modrow and coworkers [33] predict in a computer-assisted analysis of env prorein sequences of seven human immunodeficiency virus isolates antigenic epitopes in conserved and variable regions. They could identify only two antigenic sites in gp41. One of the epitopes, amino acids 722-743 probably represent the hydrophilic tail which should be located inside the cell. This sequence is followed by a more hydrophobic stretch of amino acids, which is likely to be the transmembrane region of gp41. The possible localization of the binding sites of our MAbs on an amino acid sequence (732-759) inside the cell membrane beginning with the transmembrane spanning region 3 (TM3) [33] is supported by the observation that the MAbs do not stain HIV-l-infected cells with intact cell membrane, but only cells with acetone-perforated membranes. It is noteworthy that MAbs 1Cll and 2C7, although they recognize an epitope between amino acids 732 and 759, could not neutralize HIV-1 infection in vitro (neutralization assay was performed at the Chester Beatty Laboratories, London). In contrast, Chanh and coworkers induced anti-HIV antibodies with neutralizing capacity in rabbits by a synthetic peptide corresponding to amino acid sequence 735-752 [34] and Dalgleish and coworkers demonstrated that MAbs raised against this region of gp41 could neutralize different HIV-1 strains [35]. McKeating and Willey [21] described results obtained by dif-

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ferent authors [17-20], which give evidence that broadening of the neutralizing antibody response observed as HIV infection progresses could be elicited by antibodies directed against amino acids 735-752 of gp41. We further observed that, in addition to HTLV I I I / B strain infected H9 cells, our MAbs recognized also HTLV l l l / R f and HTLV l l l / P h strain infected H9 cells (kindly provided by Dr. R. Kurth, Panl-Ehrlich-lnstltut, Frankfurt, F.R.G.) and I I I / B (Gallo) in immunofluorescence assay. In parallel to the two MAbs 1Cll, 2C7 produced in our laboratory we tested the mouse MAb M25 (Fig. 2B) and the human MAb 31)6 against 81>41 (Fig. 2C). The MAb M25, based on our data, seems to be directed exactly against the hydrophobic transmembrane region of 81)41 from set647 to pro731 [4]. This is in perfect agreement with the conclusions of diMarzo Veronese and coworkers based on a totally different approach. The fact

that human MAb 3D6 reacted with pMB255 suggests that it recognized epitopes between ile474 and trp646 of 81)160. At present it cannot be explained why 3D6 did not react with pMB1790. As the human MAb unambiguously recognizes 81>41 isolated from virions in WB analysis, one can conclude that the epitopes recognized by the antibody are located at the carboxyterminal site of the proteolyti¢ cleavage site (argS18-alaS19) between alaS19 and trp646. According to the model of Modrow and coworkers this MAb could recognize the second antigenic site from amino acids 612635. This region of the env shows homology with immunosuppressive sequences in other retroviruses, such as murine leukemia virus (MuLV) [23] and therefore MAb 3D6 could be interesting for further studies in this context. A pool of human sera containing antibodies to HIV-1 showed reactivity (in a dilution 1:100) with all fusion proteins except those encoded by pMB256, pMB254 and pMB$80. The presence of antibodies against 81>120 in these sera could be shown in WB prepared with complete HIV-1. We have previously shown [3] that the polypeptide stretches encoded by these plasmids are of less relevance for immunodiagnostie analysis. Patient sera were identified that clearly recognized recombinant env fusion proteins, especially the HIV-1 specific sequences encoded by plasmid pMB261, but which were not reactive with hybrid proteins specified by the vectors pMB256, pMB2.54 a n d / o r pMB580. Probably, the missing 81ycesylation of the recombinant 81>120 specific fusion proteins by E. coli is responsible for the poor recognition of these constructs by HIV-positive ser~t. In conclusion, with the help of this set of ten distinct recombinant HIV-1 env polypeptides we have been able to localize the epitopes of four MAbs. The epitopes recosniz~ by these MAbs are independent of glycosylption. Furthermore, these recombinant HIV-1 env polypeptides were useful for the characterization of polyclonal sera from patients. Summarizing we can say that the set of ten distinct recombinant HIV-1 env po~ypeptiOes has been proven to be a general tool for characterization of polyclonal sera as well as monoclonal antibodies, either from murine or human origin.

109 ACKNOWLEDGEMENTS W e t h a n k Dr. Josef Schneider, Deutsches Primatenzentrum, Gt~ttingen, F . R . G . , for RiPa n a l y s i s , a n d M s . B l i s a b e t h P l a n g g e r for excellent t e c h n i c a l assistance.

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