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Journal o f General Virology (1992), 73, 2451-2455. Printed in Great Britain

Inhibition of viral replication by monoclonal antibodies directed against human immunodeficiency virus gp120 M. Niedrig, ~* H.-P. Harthus, 1 J. Hinkula, 2 M . BriJker, 1 H. Bickhard, ~ G. Pauli, 3 H. R. Gelderblom 4 and B. Wahren 2 l Forschung Behringwerke AG, Postfach 1140, D-3550 Marburg, Germany, 2National Bacteriological Laboratory, Stockholm, Sweden, 3AIDS-Zentrum and 4Robert Koch-Institut des Bundesgesundheitsamtes, Nordufer 20, D-IO00 Berlin 65, Germany

Monoclonal antibodies (MAbs) were raised against the glycoprotein gp 120 of human immunodeficiency virus type 1 (strain HTLV-1IIB). The reactivity of five selected MAbs was characterized in several tests: ELISA, immunostaining of Western blots, immunofluorescence, immunoprecipitation, immunoelectron microscopy, alkaline phosphatase-anti-alkaline phosphatase assay and neutralization. The binding region was delimited by sequential overlapping Escherichia coli fusion proteins of the gpl20 sequence between amino acids (aa) 49 and 280. In the ELISA, when using

sequential overlapping 15 aa peptides, the binding epitopes were localized between aa 64 and 78 for three MAbs and between aa 114 and 123 for the fourth Mab. The fifth Mab showed multiple reactions with different peptides possibly indicating a reaction with a discontinuous epitope. In virus growth inhibition assays, all five MAbs inhibited the spread of HIV-1 infection in cell cultures after a single or repeated treatment at a concentration of 63 ~tg/ml of the purified MAbs. All MAbs showed low but significant neutralizing activity at concentrations of 100 pg/ml.

The surface glycoprotein gp 120 of HIV (SU) is proteolytically processed by cellular proteases from the env precursor gpl60 (McCune et al., 1988). SU forms tri- or tetrameric virus envelope knobs non-covalently connected by salt bonds to the viral transmembrane (TM; Leis et al., 1988) protein gp41 (Weiss et al., 1990). SU is the virus receptor for the cellular CD4 molecule present on the T helper subset of lymphocytes and antigen-presenting cells of the monocyte-macrophage lineage (Sattentau & Weiss, 1988). Gpl20 is a major target for immunological antiviral attack either by neutralizing antibodies or by cellular immune mechanisms (Javaherian et al., 1989; Lanzavecchia et al., 1988; Neurath et al., 1990a, b). Several regions of gpl20 have been defined to which antibodies are directed and/or by which neutralization is effected (Broliden et al., 1990; Linsley et al., 1988). The immunodominant V3 region [amino acids (aa) 303 to 319] has been analysed intensively by monoclonal antibodies (MAbs) (Matsushita et al., 1988; Durda et al., 1990; Dowbenko et al., 1988; Akerblom et al., 1990). Interest has focused also on other regions, in particular on the N-terminal part of gpl20 which seems to be important for interaction with the TM protein gp41 (Helseth et al., 1991 ; Ivevy-Hoyle et al., 1991 ; Broliden et al., 1992). MAbs reacting with this region may be of

interest for the analysis of the interaction of gpl20 with gp41 and of conserved neutralizing epitopes. Five MAbs directed to gpl20 of strain HTLV-IIIB were selected after fusion of SP2/0 cells with spleen cells of BALB/c mice immunized with the recombinant gp I20 fusion protein (pMB1790) mixed with 10% complete Freund's adjuvant (FA) for the first immunization and incomplete FA for the booster immunization (weeks 4 and 8). The recombinant protein has previously been shown to be highly reactive with sera from HIV-1infected individuals (BrSker et al., 1988). The specificity of the MAbs was analysed by ELISA with whole HIV-1, HIV-2 and simian immunodeficiency virus (SIV)mae antigens and in an immunoblot (Table 1). In immunofluorescence staining of HIV- or SIVmac-infected cells only one MAb (133/192) showed strong membraneassociated fluorescence of HIV-1-infected H9 giant cells in contrast to the uninfected control. Using four different HIV-1 isolates and one HIV-2 isolate grown in Molt4/ clone 8 cells the five MAbs reacted exclusively with HIV-1 in immunofluorescencetests. In a non-radioactive immunoprecipitation assay only one MAb (133/237) precipitated gpl20 from partially purified HTLV-IIIB. All five MAbs were reactive with HIV-l-infected H9 cells in alkaline phosphatase-anti-alkaline phosphatase

0001-0844 © 1992 SGM

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Short communication gpl60 gpl20 m gp41 V

Asp71S A~ I

AvaI XhoI SstI Bglli [ HindllI BamHI Bglll BgllI [ Haelll

Sail Asp718

I II II

BgllI

I

I

I

I

II

,lll

I I

I I

] t

I t

I I I I

(

Lys(I)[ Va1(49) Arg(280) \ Gly(473)x\ xl Ser(647) I ~l Leu(863) Met(8) Ser(281) 11e{474) I Gly(758) I Arg(548) Arg(732) Ser(759)

Reactivity with MAbs pMB2440

+

I

+

pMB1790 t pMB256 pMB580 pMB242 Fig. 1. Immunoelectron microscopy of HIV-1 strain HTLV-IIIB grown in H9 cells after incubation with MAb 133/192 and anti-mouse IgG-ferritin. Immune incubation of uninfected cells did not show any labelling with any of the MAbs. Bar marker represents 100 nm.

T a b l e 1. Reactivities o f M A b s against H I V - 1 g p l 2 0 MAb

lgG*

ELISA t

WBt

IF,

IP§

133/11 133/192 133/290 133/237 135/9

IgG1 IgGl IgG1 IgG1 IgG1

++** + + + + + + ++

++ + + + + + + ++

(+) + (+) (+) (+)

+ -

APAAPII NT¶ + + + + +

125 125 63 125 125

* Mouse IgG subclasses were determined by the Ouchterlony technique. t For the details of ELISA and Western blots, see Niedrig et al. (1992). The ELISA reactions were visualized by adding rabbit antimouse-conjugated peroxidase and the substrate, H202-o-phenylenediamine (OPD). For WB the reactions were visualized by goat antimouse-conjugated alkaline phosphatase (Dianova) and the appropriate substrate. See Niedrig et al. (1992). § Partially purified HIV-1 particles were used as antigens in immunoprecipitation tests according to Conraths et al. (1988). Proteins of the immunoprecipitates were separated by SDS-PAGE. Detection of antigen was performed by high titre human sera reacting with HIV-1 and anti-human IgG coupled to alkaline phosphatase (Dianova). II See Niedrig et al. (1992). ¶ Level of MAb concentration (ng/well) giving 5 0 ~ neutralization. The virus supernatant of strain HTLV-IIIB (2000 TCIDs0/ml) was incubated for 1 h at 37 °C with an equal volume of serial twofold dilutions of purified MAbs. Jurkat CD4 + (106/ml) cells suspended in RPMI-1640 culture medium with 10~ inactivated foetal calf serum a n d 2 gg/ml polybrene were incubated with the virus/MAb mixtures and seeded into four individual microtitre wells (100 gl/well). After 3 days 5 0 ~ of the culture medium was removed and fresh medium without polybrene was added. After 2 to 3 weeks culture medium was concentrated with PEG and tested for RT, as previously described (Gregersen et al., 1988). Cultures with either RT activity or giant cell formation were considered positive, cultures with neither parameter were considered negative. * * + + , Strong reaction (in ELISA .'1497 > 0-4); + , positive reaction; (+), weak positive reaction; - , negative reaction.

1

!

+

I I

I I

Fig. 2. Reaction of MAbs with the overlapping fusion proteins expressed in Escherichia coli in immunoblots. The open boxes indicate the expressed portions of the gpl60 from HIV-1 (HLTV-III, clone BH10). The gpl60 sequences were cloned in-frame to lacZ of the expression vector pBD2 (Br6ker, 1986; Br6ker et al., 1988). Cell extracts were separated by 10~ SDS-PAGE. The immunoreactions were visualized by using alkaline phosphatase-coupled anti-mouse IgG and the appropriate substrate naphthol AS-MX phosphate (Sigma). The linear peptide was synthesized according to the published eDNA sequence of Ratner et aL (1985) by a method described by Houghten (1985). Numbering of the amino acids was taken from the Los Alamos ' database (Myers et aL, 1988). The arrow indicates the cleavage site of gpl60.

(APAAP) tests, showing their usefulness in immunohistological staining. No cross-reactions with HIV-2 or SIVmac could be observed in any of the tests carried out. The immunological characterization is summarized in Table 1. The anti-gpl20 MAbs were tested for binding to the virion by pre-embedding immunoelectron microscopy (Gelderblom et al., 1987). By using indirect immunoferritin labelling, one MAb (133/192) revealed a defined label whereas the other four MAbs showed only weak SU labelling (Fig. 1). The scarce labelling might be due to the loss of gpl20 from the virion during maturation (Gelderblom et aL, 1985; Gelderblom, 1991). The binding sites of the MAbs were mapped more precisely by immunoblot analysis using a panel of overlapping recombinant fusion proteins (Fig. 2). Because all five MAbs reacted exclusively with the fusion proteins encoded by pMB2440, pMBI790 and pMB256, and no reactions were observed with pMB242 and pMB580, the binding sites were deduced to be located on the aa sequence encoded by the 231 bp Asp718/BgllI fragment (Fig. 2). To localize the binding sites, sequential overlapping synthetic peptides consisting of 15 aa covering this

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Table 2. Reactions of MAbs with 15 amino acid oligopeptides

MAb 133/11 133/237

133/290

135/9 133/192

Reference no. of coated peptides G223 G224 G225 G223 G224 G225 G227 G223 G224 G225 G227 G231 G231 G232 G214 G229 G234 G241 G249 G251 G252

Reaction Starting in positiont ELISA~/

Amino acid sequence* KAYDTEVHNVWATHA EVHNVWATHACVPTD WATHACVPTDPNPQE KAYDTEVHNVWATHA EVHNVWATHACVPTD WATHACVPTDPNPQE PNPQEVVLVNVTENF KAYDTEVHNVWATHA EVHNVWATHACVPTD WATHACVPTDPNPQE PNPQEVVLVNVTENF DMVEQMHEDI I SLWD DMVEQMHEDI I SLWD MHEDI I SLWDQSLKP GWRWGTMLLGMLMIC VVLVNNVTENFNMWKN CSLKPCVKLTPLCVS MEKGE IKNCSFNI ST TSYTLTSCNTSVI TQ SVI TQACPKVSFEP I ACPKVSFEP I P IHVC

5%73 ~78 69-83 59~3 ~78 6~83 7943 5%73 ~78 69-83 7943 10~123 109-123 11~128 8-23 8~1~ 123-137 163-177 203-217 213-227 218-232

2.0 2-1 1.9 1-9 2.0 2-0 0.7 2.0 2.0 2.0 0-7 1.1 1-9 2.0 0.65 0.85 0.8 1.3 1.0 0.75 0-8

* Overlapping amino acid sequences are underlined. I"Starting positions were taken from the database (Myers et al., 1988). :~The dilution of antibody corresponding to an A497 of 0-6 to 1.2 in the HIV-1 ELISA was used for incubation with different peptides coupled to microtitre plates. The reactions were visualized by adding anti-mouse IgG-conjugated peroxidase and H202-OPD. The mean values for background reactivity (Aa97) with other irrelevant p24 peptides were < 0.25. Values > 0-5 were considered as specific reactions.

region were used (Akerblom et al., 1990). Each subsequent peptide overlapped the previous one by 10 aa. In ELISA plates coated with these peptides the MAbs 133/11, 133/290 and 133/237 reacted with three overlapping peptides (Table 2). We concluded that the epitope is represented by aa 64 to 78 and that M A b 135/9 which reacted with two peptides covers the epitope aa 114 to 123. M A b 133/192 reacted with seven different peptides, two of them overlapping each other. It seems that this M A b reacts with a discontinous epitope represented by more than two binding sites. The E L I S A results were confirmed by a competition assay using peroxidase-conjugated MAbs. The three MAbs 133/11, 133/290 and 133/237 inhibited each other, indicating the recognition of the same binding sites. As expected the two other M A b s did not interfere with any other MAb, clearly showing that they react with different epitopes. In a cell culture system the M A b s were analysed for antiviral activity. HIV-l-infected C D 4 ÷ Jurkat cells were mixed with uninfected cells at a ratio of 1 : 10 and seeded in 96 wells. Four wells were treated once, four other wells additionally on days 3 and 7 with different concentrations (32 to 500 ~tg/ml) of purified anti-gpl20

MAbs. Two anti-p24 and one anti-pl7 MAbs described previously were used as controls (Niedrig et al., 1988, 1989). Cultures were monitored microscopically and by analysis of reverse transcriptase (RT) activity in the culture supernatant in a microassay (Gregersen et al., 1988). The R T activity was calculated as a percentage relative to the untreated cell culture (Fig. 3). After single or multiple treatments with high concentrations of the five anti-gpl20 MAbs all HIV-l-infected cell cultures showed a strong inhibition of virus spread over a period of 3 weeks (Fig. 3). Using a lower concentration of antigpl20 MAbs (32 ~tg/ml) the antiviral effect was reduced, showing the concentration-dependent effect of the antibodies. The anti-p24 and anti-pl7 M A b s showed no antiviral activity under the same conditions (63 ~tg/ml) compared to the anti-gpl20 MAbs. Only under repeated treatments with high concentrations (500 ~tg/ml) of purified anti-p24 or anti-pl7 M A b could a decrease in RT activity to 20% be observed. After stopping the treatment with anti-p24 or anti-pl7 MAbs, R T activity increased up to 80% within 10 days (data not shown). The epitopes represented by aa 64 to 78 and aa 114 to 123 belong to a conserved region located in the Nterminal part of gpl20 (Modrow et al., 1987). MAbs

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i

i

i

i

i

providing HIV and SIV strains and susceptible cells. We also thank S. Modrow, Munich, Germany for providing gpl20 peptides and helpful discussions.

100 80 t-

References

60 40 20 0

~ 3

-

7

10 14 Time (days)

II 21

Fig. 3. Effect of repeated treatment of fresh HIV-l-infected cells with purified MAbs. The cultures were supplemented with 63 ktg/ml of the following antibodies: (X7) 133/11, (©) 133/192, ( x ) 133/290, (+) 133/237, (11) 135/09 anti-p24; (V) 8-D-2, (&) 12-B-4, anti-pl7; (n) 3-H-7 as described previously (Niedrig et al., 1988, 1989). RT (%) represents percentage of RT activity compared to the untreated HIV-1infected cell culture control. RT activity was determined in the supernatant of treated and untreated HIV-1-infected cell cultures by a microscale assay according to Gregersen et al. (1988). The arrows indicate the time points at which medium containing 63 ~g/ml of the different antibodies was added.

described previously by Akerblom et al. (1990) which bind close to the epitopes described here do not neutralize HIV-1 according to these authors. This finding contrasts with our results where we found low although significant neutralization activity as well as strong antiviral activity. From predicted protein structures the gpl20 epitopes described should be accessible to antibodies (Modrow et al., 1987; Starcich et al., 1987); we could show by immunoelectron microscopy that viruses were labelled by the antibodies. In sera from HIV-1- and HIV-2-infected individuals, antibodies were found in a low percentage (12 to 309/0) reacting with a peptide representing the epitope spanning aa 64 to 78 and 35 % recognizing the region of aa 119 to 123 of gp 120 (Norrby et al., 1991). This conserved region of gpl20 does not seem to be immunodominant in man. Whether an immune response against these epitopes might influence the course of the infection is not known. Our data on the neutralization and growth inhibition suggest that this part of the protein might play a crucial role in the cell-to-cell spread of the virus. Inclusion of these epitopes together with peptides including a strong neutralizing immune response into a post-infection vaccine might increase the protective immune response against HIV. The authors thank Mrs S. Mehdi, L. Hopp and Mr S. Hahn for excellent technical assistance, Mrs R. Blesken, G. Kulins and H. Oldenburg for performing the APAAP immunocytochemistry, and Mr H. Reupke for immunoelectron microscopy. We are grateful to R. Desrosiers, Southborough, Mass., U.S.A., R. C. Gallo and M. Popovic, Bethesda, Md., U.S.A., and L. Montagnier, Paris, France for

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(Received 13 January 1992; Accepted 1 May 1992)