VIROLOGY
175,600-604
(1990)
Complement-Mediated Antibody-Dependent Enhancement of HIV-1 Infection Requires CD4 and Complement Receptors’ W. EDWARD ROBINSON, JR., DAVID C. MONTEFIORI, AND WILLIAM M. MITCHELL Department
of Pathology,
Vanderbilt University,
Nashville,
Received September 25, 1989; accepted December
Tennessee 37232 27, 1989
In this study it is demonstrated that complement-mediated antibody-dependent enhancement (C’-ADE) of HIV-1 infection in vitro is blocked by murine monoclonal antibodies to CD4 and complement receptor type 2 (CR2) while HIV1 infection in the absence of C’-ADE is blocked by anti-CD4 but not anti-CR2 monoclonal antibodies. The anti-CR2 murine monoclonal antibody, OKB7, blocked C’-ADE of HIV-1 infection at concentrations greater than 1 pglml. The anti-CD4 monoclonal antibody, OKT4a, but not OKT4f blocked C’-ADE at concentrations greater than 0.06 pg/ml. HIV1 infections were quantitated by cytopathic effect, indirect immunofluorescence, and reverse transcriptase release. It appears from these in vitro studies that C’-ADE of HIV-1 infection requires both CD4 and complement receptors while HIV-1 infection in the absence of antibody and complement requires only CD4. o 199OAcademic PWSS. h.
Homsy et a/. have recently shown that FcR-ADE of HIV infection required Fc receptor type 2 but not CD4 (20). In contrast, other investigators have reported that FcR-ADE of HIV infection required both CD4 and Fc receptors on monocytes and macrophages (F. Jouault et al., Abstract No. 2083, IV Int’l Conference on AIDS, Stockholm, Sweden, 1988; S. Matsuda et al., Abstract No. 2070, IV Int’l Conference on AIDS; M. Zeira et al., Abstract No. W.C.P. 135, V Int’l Conference on AIDS, Montreal, Quebec, Canada, 1989; B. Zorr et a/., Abstract No. W.C.P. 66, V Int’l Conference on AIDS). In this study, we demonstrate that C’ADE of HIV-l infection requires coexpression of CD4 and CR2 since C’-ADE was blocked by both a murine monoclonal antibody to CR2 and murine monoclonal antibodies to CD4. For all C’-ADE assays, serum No. 200 was used as the enhancing serum. Serum No. 200 is a control enhancing serum that has been extensively characterized (Table 1). The C’-ADE titer, anti-HIV-l titer in the presence and absence of complement, and the anti-syncytial titer of this serum are given in Table 1. Serum No. 200 is representative of the over 100 human sera that have been assayed for C’-ADE of HIV-1 infection. It enhances HIV-l infection to high titer (1: 16,200 dilution). Further, it has a significant HIV-1 neutralizing titer (1:256) in the absence of complement which is lowered by the addition of complement (1:40). As with most HIV-1 antibody-positive sera, it has a low anti-syncytial titer (1:80). This serum is similar to the other HIV-l antibody-positive sera we have tested as approximately 30% have similar C’-ADE (1 :10,000-l :20,000), anti-
The acquired immune deficiency syndrome (AIDS) has been associated with severe, unrelenting, opportunistic infections and rare neoplasms in humans secondary to failure of the immune system. A significant body of evidence has shown the human immunodeficiency virus (HIV) to be the causative agent in AIDS. Although it is generally believed that some antibodies to HIV are neutralizing and, therefore, should prevent disease progression (I-4), no anti-HIV vaccines have been found to protect chimpanzees, the only animal model of HIV infection, from subsequent virus challenge (5). Further, passive transfer of high quantities of neutralizing antibody to chimpanzees failed to protect those chimpanzees from subsequent challenge with HIV (6). One of several possible explanations for vaccine failure is antibody-dependent enhancement (ADE) of HIV infection. ADE of virus infectivity is a well-described phenomenon (7-11) and has been demonstrated for HIV-1 (12-20). Two mechanisms for ADE of HIV-1 infection have been demonstrated in vitro: the first to be described required antibody to HIV1 envelope glycoproteins and the alternative pathway of complement (12- 16); the second required antibody to HIV-l and was shown to utilize Fc receptors on monocytes, macrophages, and lymphocytes (78, 19). To distinguish between these two mechanisms, the former has been called complement-mediated ADE (C’ADE) while the latter has been referred to as Fc receptor-mediated ADE (FcR-ADE).
’ This work was supported in pat-l by NIH Contract NOl-Al52576 and NIH Grants Al 25272 and Al29398.
0042-6822190
$3.00
Copyright 0 1990 by Academac Press. Inc. All rights of reproduction in any form reserved
600
601
SHORT COMMUNICATIONS TABLE 1 C’-ADE, ANTI~YNCMIAL, AND ANTI-HIV-l TITERSOF SERUM No. 200a Anti-HIV-lb
C’-ADE”
Anti-HIV-l Induced syncytiad
1: 16,200
1:80
Minus complement 1:256
Plus complement 1:40
a Serum No. 200 was from an HIV-1 antibody-positive donor of unknown clinical stage. ‘The anti-HIV-l activity of heat-inactivated serum was determined against cellfree virus in the presence and absence of 1:20 human complement as described ( 17,33). Titer is the greatest dilution giving >50% protection from cytopathic effect. c C-ADE titer was determined as described (17). Threefold dilutrons of heat-inactivated serum were added to 1:20 human complement in triplicate. Cellfree HIV-1 was then added followed by MT-2 cells. Cells were incubated for 3 days at 37” then assayed for cell viability as described (33). Titer was defined as highest dilution giving 50% more viral-induced cytopathic effect than cells treated with HIV1 and complement. Enhancement begins at the first dilution after neutralization in the presence of complement ends (i.e., 1: 120). d Anti-syncytial titer was determined by performing triplicate, twofold dilutions of heat-inactivated serum in RPMI-1640 with 12% fetal calf serum. H9 cells chronically infected with the HTLV-IlIe isolate of HIV-1 were added followed by MT-2 cells. Cells were incubated for 24 hr at 37” then assayed for viral-induced cytopathic effect as described (33). Titer is the greatest dilution giving 50% protection from cytopathrc effect.
syncytial (1:64-l :128), and neutralizing (1:256-l :512) titers (17; and unpublished results). For C’-ADE experiments reported in these studies, heat-inactivated serum No. 200 was diluted 1:500 into growth medium containing 1:20 fresh, normal human serum as a complement source as previously described (17). Although previous experiments using a murine monoclonal antibody to CR2, anti-CR2 (Becton-Dickinson), had failed to block C’-ADE of HIV-l infection (17) an anti-CR2 monoclonal antibody, OKB7, recognizing a different epitope on CR2, was used in the present study. This second murine monoclonal antibody, OKB7, has been shown to block two functional activities of CR2 (21-25). The activities reportedly blocked by OKB7 include binding of both complement component C3d,g (21, 22) and Epstein-Barr virus (21-25) to CR2. The previously studied anti-CR2 monoclonal antibody (17) does not block the functional activity of CR2. As Fig. 1 shows, OKB7 could block C’-ADE of HIV-1 infection at concentrations greater than 1 pg/ml. Cell viability was relative to a HIV-l plus 1:20 complement control (100% viable) while C’-ADE control was 0% viable. A control murine monoclonal antibody, OKT4f, had no activity in this assay while OKT4a blocked C’-ADE completely at concentrations greater than 0.06 pg/ml
(see Table 2). Thus, by viral-induced cytopathic effect, C’-ADE was blocked by OKB7. To determine whether CD4 was required for C’-ADE, several murine anti-CD4 monoclonal antibodies were tested for their anti-HIV, anti-syncytia formation, and anti-C’-ADE activities. As shown in Table 2, OKT4a blocked C-ADE, HIV-1 infectivity, and HIV-1 -induced syncytia formation at concentrations from 0.06 to 0.4 pg/ml (Table 2). OKT4f, an anti-CD4 monoclonal antibody that has no anti-HIV-l activity in this laboratory, failed to block any aspect of HIV-l infection even at concentrations of 55 pg/ml. OKNK, a control IgM that does not bind to MT-2 cells (see Table 2), also failed to block any aspect of HIV-1 infection at concentrations as high as 50 pglml (Table 2). These data demonstrate that anti-CD4 monoclonal antibodies which block HIV1 infection also block C’-ADE of HIV-1 infection in vitro. To confirm that the anti-HIV and anti-C’-ADE activities of murine monoclonal antibodies were not determined by differential expression of antigens on Ml-2 cells, flow cytometric analysis was performed. As shown in Table 2, OKB7, OKT4a, and OKT4f all bind to MT-2 cells. The OKNK epitope is not expressed on MT2 cell surfaces. Therefore, the inability of OKT4f to inhibit C’-ADE was not because the OKT4f epitope is not expressed on MT-2 cells but rather because that epitope is not involved in either C’-ADE or HIV-l infection. Likewise, the failure of OKB7 to block C’-ADE to the same extent as OKT4a was not due to a major difference in the number of cells expressing the OKB7 epitope. Having demonstrated that C’-ADE of HIV-1 infection was blocked by anti-CD4 and anti-CR2 monoclonal antibodies using virus-induced cytopathic effect as an indicator of HIV-1 infection (Table 2 and Fig. l), the anti-
0-0
10 20 Antibody Concentration
FIG. 1. OKB7 protection of MT-2 cells tron. 100% viability control represents cells challenged with HIV-1 ; 0% viability eight replicates of MT-2 cells challenged complement and 1:500 heat-inactivated trol). Final concentrations of OKB7 were
30
40
(#g/ml)
from C-ADE of HIV-1 infecmean of eight replicates of control represents mean of with HIV-l plus 1:20 human serum No. 200 (C’-ADE con35-0.5 pg/ml.
SHORT COMMUNICATIONS
602
TABLE 2 ANTI-HIV-~ ACTIVITIESOF MURINE MONOCLONALANTIBODIES Anti-HIV-l
Monoclonal
antibody
OKT4a OKT4f OKNK OKB7
Plus C’ADE’ kh-4
Minus C’-ADE* Wml)
0.06 NI” NI 1
0.20 NI NI NI
Percentage of epitope positive MT-2 cellsd
Anti-syncytiac Wml) 0.40 55 NI NI
96 81 0 90
BAnti-HIV-l plus C’-ADE titer was determined in the presence of 1:500 serum No. 200 and 1:20 human complement and refers to the concentration of antibody required to give >50% protection of cells from C’-ADE of HIV-1 infection. See Table 1. b Anti-HIV-l activity minus C’ADE was determined in the absence of complement as described in Table 1 and refers to the concentration of antibody required to give ~50% protection from HIV-l-induced cytopathic effect. c Anti-syncytia titer was determined as in Table 1 and refers to the concentration of antibody required to give >50% protection from HIV-1 induced syncytia formation after HIV-1 chronically infected H9 cells were mixed with uninfected MT-2 cells. d Flow cytometry using indirect immunofluorescence of unfixed MT-2 cells was performed on a Coulter EPICS 753 multiparameter flow cytometer. Control IgM and IgG were subtracted from values prior to calculation of percent positive cells. s No inhibition at any concentration tested. Highest concentration of all monoclonal antibodies tested was 50-60 pg/ml.
C’-ADE activity was confirmed using other parameters of HIV-l infection. As illustrated by Table 3, C’-ADE of HIV-l infection was blocked by both anti-CD4 and antiCR2 murine monoclonal antibodies when either HIV-l immunofluorescent (IF) positive cells or reverse transcriptase (RT) release was used as a marker of HIV-1 infection. When C’-ADE was assayed in the presence of 60 pg/ml of OKB7, only 50% of cells expressed HIV-
1 antigens compared to 100% of cells treated with enhancing antibody and OKNK or OKT4f at the same concentration. Cells challenged with virus and complement alone (no C’-ADE) were also 100% positive for HIV-1 antigen. OKT4a blocked C’ADE significantly as only 5% of cells were positive for HIV-1 antigens. When progeny virions were quantitated using RT release, OKB7 reduced the amount of RT from 2,105,OOOcpm/
TABLE 3 ANTI-HIV-~ ACTIVIN OF MURINE MONOCLONAL ANTBODIES CONFIRMEDBY PERCENTAGEANTIGEN POSITIVECELLS AND RT RELEASEFROM HIV-1 -CHALLENGED MT-2 CELLS Plus C’-ADEB
Monoclonal
antibody
OKNK OKT4a OKT4f HIV + Compl’ OKB7
Percentage IF positive cellsc looe 5 100 100 50
Minus C’-ADEb RT (cpm/ml)d
2,105,OOO 14,824 667,440 492,536 167,544
f 103,888 + 10,560 + 126,848 + 24,168 f 5,656
Percentage IF positive cells
RT (cpm/ml)
100 0 100 NTQ NT
2,748,773 + 169,268 13,453 + 9,840 1,494.960 + 100,277 NT NT
BAnti-HIV activity plus C’-ADE was determined in the presence of diluted (1:500) serum No. 200 and 1:20 human complement. Virus and antibodies were removed 24 hr after initial virus challenge. Results are for 72 hr after initial virus challenge. All monoclonal antibodies were tested at 60 pg/ml. * Anti-HIV activity minus C’-ADE was determined for the HTLV-Ills isolate of HIV-l. Virus and antibodies were removed 24 hr after initial virus challenge. Results are for 72 hr after the initial virus challenge. All monoclonal antibodies were tested at 60 pg/ml. c IF positive cells were quantitated using polyclonal human anti-HIV-l serum and FITC-conjugated goat anti-human IgG (Cappel) as described (34). d Reverse transcriptase release was quantitated using the method of Poiesz eta/. (3% ’ Denotes lysis of culture. ‘ HIV + 1:20 human complement and no enhancing antibody. Q Not tested.
603
SHORT COMMUNICATIONS
ml (OKNK) to 167,544 cpm/ml (Table 3). OKT4f had some anti-C’-ADE activity reducing RT release approximately 3-fold. OKT4a reduced RT release by greater than lOO-fold. HIV-l plus complement yielded 4-fold less RT release than the C’-ADE control. OKB7 had a greater effect on RT and HIV-1 antigen positive cells than the lack of enhancing antibody (complement plus virus alone) not because it had any anti-HIV activity but rather because dilute complement (1:40) alone has been shown to have weak HIV-1 infection-enhancing activity (16, 17). The anti-HIV-l activity of these monoclonal antibodies was similar whether tested in MT-2 cells in the absence of C’-ADE (Table 3) or in H9 cells (data not shown). Values of RT and IF positive cells cannot be compared for plus C’-ADE and minus C’-ADE in Table 3 because they are from two independent experiments. The data presented herein have shown that C’-ADE of HIV-1 infection in MT-2 cells requires complement receptors and CD4. The specific complement receptor required for C’-ADE of HIV-1 infection in MT-2 cells is CR2. It is possible that other complement receptors on other cells might also serve as receptors for C’-ADE of HIV-1 infection. CD4 was also required for both HIV1 infection and C’-ADE of HIV-1 infection as OKT4a, a potent anti-HIV-l monoclonal antibody (26-32), had both anti-HIV-1 and anti-C’-ADE activities. Although the exact role of complement and antibody to HIV-l in the natural pathogenesis of HIV-1 remains to be determined, it is apparent that cells bearing either Fc or complement receptors can be targets for HIV-l. The mechanism by which CR2 and complement enhance HIV-1 infection in vitro is unknown. However, HIV-1 opsonized with complement and anti-envelope antibodies may bind to cells with a higher affinity than nonopsonized virus binding to CD4 alone. Likewise, binding to CR2 may aid in virus penetration or uncoating or may cause alternative intracellular routing of HIV-l, thereby leading to increased replication efficiency as was hypothesized previously ( 17). Whatever the mechanism by which complement enhances infection in vitro, its in viva relevance is not understood. However, since the addition of complement can significantly reduce or completely abrogate the in vitro protective effects of HIV-l neutralizing antibodies present in heat-inactivated serum, it is imperative that the role of ADE in HIV1 infection in viva be determined. ACKNOWLEDGMENTS The authors thank Cecilia Haberzettl and William Covert of Ottho Diagnostics. Raritan, NJ, for the generous gift of murine monoclonal antibodies OKT4a. OKT4f, OKNK. and OKB7 used in these studies.
Critical discussions of the work were graciously provided by Drs. Tamar Ben-Porat. David Karzon. and Clark Tibbetts.
REFERENCES 1. WEISS, R. A., CLAPHAM, P. R., CHEINGSONG-POPOV.R.. DALGLEISH, A. G.. CARNE, C. A., WELLER, I. V. D., ~~~TEDDER. R. S., Nature (London) 316, 69-72 (1985). 2. ROBERT-GUROFF,M., BROWN, M., and GALLO, R. C., Nature (London) 316, 72-74 (1985). 3. RASHEED,S., NORMAN, G. L., GILL, P. S., MEYER, P. R., CHENG, L., and LEVINE,A. M., Virology 150, l-9 (1986). 4. WEISS, R. A., CLAPHAM, P. R., WEBER, J. N.. DALGLEISH, A. G., LASKY, L. A., and BERMAN, P. W., Nature (London) 324, 572574 (1986). 5. BERMAN, P. W., GROOPMAN, J. E., GREGORY,T., CLAPHAM, P. R., WEISS, R. A., FERRIANI,R., RIDDLE, L., SHIMASAKI,C., LUCAS, C., LASKY, L. A., and EICHBERG.J. W., Proc. Narl. Acad. SC;. USA 85,5200-5204 (1988). 6. PRINCE, A. M., HOROWIZ. B.. BAKER, L., SHULMAN, R. W., RALPH, H., VALINSKY,J.. CUNDELL, A., BROTMAN, B., BOEHLE, W., REY, F., PIET, M.. REESINK.H., LELIE, N.. TERSMETTE,M., MIEDEMA, F., BARBOSA, L., NEMO, G.. NASTALA, C. L., ALLAN, J. S., LEE, D. R., and EICHBERG,J. W.. Proc. Nat/. Acad. SC;. USA 85, 6944-6948 (1988). 7. PORTERFIELD,J. S., Adv. Virus Res. 31,335-355 (1986). 8. MITCHELL, W. M., ROBINSON, W. E., JR., and MONTEFIORI, D. C., Infect. Dis. Newsleft. 7, 59-60 (1988). 9. HALSTEAD, S. B., Science 239,476-481 (1988). 10. CARDOSA, M. J., PORTERFIELD,J. S., and GORDON, S., J. fxp. Med. 158,258-263(1983). 11. KLIKS, S. C., NISALAK. A., BRANDT, W. E., WAHL, L., and BURKE, D. S., Amer. J. Trop. Med. Hyg. 40,444-451 (1989). 12. ROBINSON, W. E.. JR., MONTEFIORI, D. C., and MITCHELL, W. M., 149,693-699 (1987). 13. ROBINSON, W. E., JR., MONTEFIORI, D. C., and MITCHELL, W. M., Lancet i, 790-794 (1988). 14. ROBINSON, W. E., JR., MONTEFIORI, D. C., and MITCHELL, W. M., Lancet i, 830-831 (1988). 15. GRAS, G., STRUB. T., and DORMONT, D., Lancet i, 1285 (1988). 16. ROBINSON, W. E., JR., MONTEFIORI, D. C.. MITCHELL, W. M., PRINCE, A. M., ALTER, H. J., DREESMAN, G. R.. and EICHBERG, J. W., Proc. Nat/. Acad. Sci. USA 86, 471 O-4714 (1989). 17. ROBINSON, W. E., JR., MONTEFIORI, D. C., GILLESPIE, D. H., and MITCHELL, W. M., J. Acquiredlmmune Defic. Syndrome 2,3342 (1989). 18. HOMSY, J., TATENO, M., and LEVY, 1. A., Lancet i, 1285-1286 (1988). 19. TAKEDA, A., TUAZON, C. U., and ENNIS. F., Science 242, 580-583 (1988). 20. HOMSY, J., MEYER, M., TATENO, M., CLARKSON,S., and LEVY, 1. A., Science 244, 1357-l 360 (1989). 21. MOLD, C., NEMEROW. G. R., BRADT, B. M., and COOPER, N. R., /. lmmunol. 140, 1923-1929(1988). 22. LAMBRIS, J. D., GANU, V. S., HIRANI, S., and MOLLER-EBERHARD, H. I., Proc. Nat/. Acad. SC;. USA 82, 4235-4239 (1985). 23. NEMEROW.G. R., WOLFERT, R., MCNAUGHTON. M. E.. and COOPER, N. R.,/. Viral. 55, 347-351 (1985). 24. NEMEROW. G. R., MOLD, C., SCHWEND, V. K., TOLLEFSEN,V.. and COOPER, N. R.,/. Virol.61, 1416-1420(1987).
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25. NEMEROW, G. R., HOUGHTEN, R. A., MOORE, M. D., and COOPER, N. R., Cell56, 369-377 (1989). 26. MCDOUGAL, J. S., MAWLE, A., CORT, S. P., NICHOLSON, J. K. A., CROSS,G. D., SCHEPPLER-CAMPBELL,J. A., HICKS, D., and SLIGH, J.,J./mmunol. 135,3151-3162(1985). 27. MCDOUGAL, J. S., KENNEDY, M. S., SLIGH, J. M., CORT, S. P., MAWLE, A., and NICHOLSON, 1. K. A., Science 231, 382-385 (1986). 28. MCDOUGAL, J. S., NICHOLSON, J. K. A., CROSS, G. D., CORT, S. P., KENNEDY,M. S., and MAWLE, A., J. Immunol. 137,2937-2944 (1986). 29. MATHEWS, T. J., WEINHOLD, K. J., LYERLY,H. K., LANGLOIS,A. J., WIGZELL, H., and BOLOGNESI,D. P., Proc. Natl. Acad. Sci. USA 84,5424-5428 (1987). 30. LASKY. L. A., NAKAMURA, G., SMITH, D. H.. FENNIE, C., SHIMASAKI,
31. 32.
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C., PATZER,E., BERMAN, P., GREGORY,T., and CAPON, D. J., Cell 50,975-985 (1987). MIZUKAMI, T., FUERST,T. R., BERGER,E. A., and Moss, B., Proc. Natl. Acad. Sci. USA 85, 9273-9277 (1988). JAMESON, B. A., RAO, P. E., KONG, L. I., HAHN, 8. G., SHAW, G. M., HOOD, L. E., and KENT, S. B. H., Science 240, 13351339 (1988). MONTEFIORI, D. C., ROBINSON, W. E., JR., SCHUFFMAN, S. S., and MITCHELL, W. M., J. C/in. Microbial. 26, 231-235 (1988). MONTEFIORI, D. C., and MITCHELL, W. M., virology 155,726-731 (1986). POIESZ, B. J.. RUSCEITI, F. W.. GAZDAR, A. F., BUNN, P. A., MINNA, J. D., and GALLO, R. C., Proc. Nat/. Acad. Sci. USA 77, 7415 7419 (1980).
175,600-604
(1990)
Complement-Mediated Antibody-Dependent Enhancement of HIV-1 Infection Requires CD4 and Complement Receptors’ W. EDWARD ROBINSON, JR., DAVID C. MONTEFIORI, AND WILLIAM M. MITCHELL Department
of Pathology,
Vanderbilt University,
Nashville,
Received September 25, 1989; accepted December
Tennessee 37232 27, 1989
In this study it is demonstrated that complement-mediated antibody-dependent enhancement (C’-ADE) of HIV-1 infection in vitro is blocked by murine monoclonal antibodies to CD4 and complement receptor type 2 (CR2) while HIV1 infection in the absence of C’-ADE is blocked by anti-CD4 but not anti-CR2 monoclonal antibodies. The anti-CR2 murine monoclonal antibody, OKB7, blocked C’-ADE of HIV-1 infection at concentrations greater than 1 pglml. The anti-CD4 monoclonal antibody, OKT4a, but not OKT4f blocked C’-ADE at concentrations greater than 0.06 pg/ml. HIV1 infections were quantitated by cytopathic effect, indirect immunofluorescence, and reverse transcriptase release. It appears from these in vitro studies that C’-ADE of HIV-1 infection requires both CD4 and complement receptors while HIV-1 infection in the absence of antibody and complement requires only CD4. o 199OAcademic PWSS. h.
Homsy et a/. have recently shown that FcR-ADE of HIV infection required Fc receptor type 2 but not CD4 (20). In contrast, other investigators have reported that FcR-ADE of HIV infection required both CD4 and Fc receptors on monocytes and macrophages (F. Jouault et al., Abstract No. 2083, IV Int’l Conference on AIDS, Stockholm, Sweden, 1988; S. Matsuda et al., Abstract No. 2070, IV Int’l Conference on AIDS; M. Zeira et al., Abstract No. W.C.P. 135, V Int’l Conference on AIDS, Montreal, Quebec, Canada, 1989; B. Zorr et a/., Abstract No. W.C.P. 66, V Int’l Conference on AIDS). In this study, we demonstrate that C’ADE of HIV-l infection requires coexpression of CD4 and CR2 since C’-ADE was blocked by both a murine monoclonal antibody to CR2 and murine monoclonal antibodies to CD4. For all C’-ADE assays, serum No. 200 was used as the enhancing serum. Serum No. 200 is a control enhancing serum that has been extensively characterized (Table 1). The C’-ADE titer, anti-HIV-l titer in the presence and absence of complement, and the anti-syncytial titer of this serum are given in Table 1. Serum No. 200 is representative of the over 100 human sera that have been assayed for C’-ADE of HIV-1 infection. It enhances HIV-l infection to high titer (1: 16,200 dilution). Further, it has a significant HIV-1 neutralizing titer (1:256) in the absence of complement which is lowered by the addition of complement (1:40). As with most HIV-1 antibody-positive sera, it has a low anti-syncytial titer (1:80). This serum is similar to the other HIV-l antibody-positive sera we have tested as approximately 30% have similar C’-ADE (1 :10,000-l :20,000), anti-
The acquired immune deficiency syndrome (AIDS) has been associated with severe, unrelenting, opportunistic infections and rare neoplasms in humans secondary to failure of the immune system. A significant body of evidence has shown the human immunodeficiency virus (HIV) to be the causative agent in AIDS. Although it is generally believed that some antibodies to HIV are neutralizing and, therefore, should prevent disease progression (I-4), no anti-HIV vaccines have been found to protect chimpanzees, the only animal model of HIV infection, from subsequent virus challenge (5). Further, passive transfer of high quantities of neutralizing antibody to chimpanzees failed to protect those chimpanzees from subsequent challenge with HIV (6). One of several possible explanations for vaccine failure is antibody-dependent enhancement (ADE) of HIV infection. ADE of virus infectivity is a well-described phenomenon (7-11) and has been demonstrated for HIV-1 (12-20). Two mechanisms for ADE of HIV-1 infection have been demonstrated in vitro: the first to be described required antibody to HIV1 envelope glycoproteins and the alternative pathway of complement (12- 16); the second required antibody to HIV-l and was shown to utilize Fc receptors on monocytes, macrophages, and lymphocytes (78, 19). To distinguish between these two mechanisms, the former has been called complement-mediated ADE (C’ADE) while the latter has been referred to as Fc receptor-mediated ADE (FcR-ADE).
’ This work was supported in pat-l by NIH Contract NOl-Al52576 and NIH Grants Al 25272 and Al29398.
0042-6822190
$3.00
Copyright 0 1990 by Academac Press. Inc. All rights of reproduction in any form reserved
600
601
SHORT COMMUNICATIONS TABLE 1 C’-ADE, ANTI~YNCMIAL, AND ANTI-HIV-l TITERSOF SERUM No. 200a Anti-HIV-lb
C’-ADE”
Anti-HIV-l Induced syncytiad
1: 16,200
1:80
Minus complement 1:256
Plus complement 1:40
a Serum No. 200 was from an HIV-1 antibody-positive donor of unknown clinical stage. ‘The anti-HIV-l activity of heat-inactivated serum was determined against cellfree virus in the presence and absence of 1:20 human complement as described ( 17,33). Titer is the greatest dilution giving >50% protection from cytopathic effect. c C-ADE titer was determined as described (17). Threefold dilutrons of heat-inactivated serum were added to 1:20 human complement in triplicate. Cellfree HIV-1 was then added followed by MT-2 cells. Cells were incubated for 3 days at 37” then assayed for cell viability as described (33). Titer was defined as highest dilution giving 50% more viral-induced cytopathic effect than cells treated with HIV1 and complement. Enhancement begins at the first dilution after neutralization in the presence of complement ends (i.e., 1: 120). d Anti-syncytial titer was determined by performing triplicate, twofold dilutions of heat-inactivated serum in RPMI-1640 with 12% fetal calf serum. H9 cells chronically infected with the HTLV-IlIe isolate of HIV-1 were added followed by MT-2 cells. Cells were incubated for 24 hr at 37” then assayed for viral-induced cytopathic effect as described (33). Titer is the greatest dilution giving 50% protection from cytopathrc effect.
syncytial (1:64-l :128), and neutralizing (1:256-l :512) titers (17; and unpublished results). For C’-ADE experiments reported in these studies, heat-inactivated serum No. 200 was diluted 1:500 into growth medium containing 1:20 fresh, normal human serum as a complement source as previously described (17). Although previous experiments using a murine monoclonal antibody to CR2, anti-CR2 (Becton-Dickinson), had failed to block C’-ADE of HIV-l infection (17) an anti-CR2 monoclonal antibody, OKB7, recognizing a different epitope on CR2, was used in the present study. This second murine monoclonal antibody, OKB7, has been shown to block two functional activities of CR2 (21-25). The activities reportedly blocked by OKB7 include binding of both complement component C3d,g (21, 22) and Epstein-Barr virus (21-25) to CR2. The previously studied anti-CR2 monoclonal antibody (17) does not block the functional activity of CR2. As Fig. 1 shows, OKB7 could block C’-ADE of HIV-1 infection at concentrations greater than 1 pg/ml. Cell viability was relative to a HIV-l plus 1:20 complement control (100% viable) while C’-ADE control was 0% viable. A control murine monoclonal antibody, OKT4f, had no activity in this assay while OKT4a blocked C’-ADE completely at concentrations greater than 0.06 pg/ml
(see Table 2). Thus, by viral-induced cytopathic effect, C’-ADE was blocked by OKB7. To determine whether CD4 was required for C’-ADE, several murine anti-CD4 monoclonal antibodies were tested for their anti-HIV, anti-syncytia formation, and anti-C’-ADE activities. As shown in Table 2, OKT4a blocked C-ADE, HIV-1 infectivity, and HIV-1 -induced syncytia formation at concentrations from 0.06 to 0.4 pg/ml (Table 2). OKT4f, an anti-CD4 monoclonal antibody that has no anti-HIV-l activity in this laboratory, failed to block any aspect of HIV-l infection even at concentrations of 55 pg/ml. OKNK, a control IgM that does not bind to MT-2 cells (see Table 2), also failed to block any aspect of HIV-1 infection at concentrations as high as 50 pglml (Table 2). These data demonstrate that anti-CD4 monoclonal antibodies which block HIV1 infection also block C’-ADE of HIV-1 infection in vitro. To confirm that the anti-HIV and anti-C’-ADE activities of murine monoclonal antibodies were not determined by differential expression of antigens on Ml-2 cells, flow cytometric analysis was performed. As shown in Table 2, OKB7, OKT4a, and OKT4f all bind to MT-2 cells. The OKNK epitope is not expressed on MT2 cell surfaces. Therefore, the inability of OKT4f to inhibit C’-ADE was not because the OKT4f epitope is not expressed on MT-2 cells but rather because that epitope is not involved in either C’-ADE or HIV-l infection. Likewise, the failure of OKB7 to block C’-ADE to the same extent as OKT4a was not due to a major difference in the number of cells expressing the OKB7 epitope. Having demonstrated that C’-ADE of HIV-1 infection was blocked by anti-CD4 and anti-CR2 monoclonal antibodies using virus-induced cytopathic effect as an indicator of HIV-1 infection (Table 2 and Fig. l), the anti-
0-0
10 20 Antibody Concentration
FIG. 1. OKB7 protection of MT-2 cells tron. 100% viability control represents cells challenged with HIV-1 ; 0% viability eight replicates of MT-2 cells challenged complement and 1:500 heat-inactivated trol). Final concentrations of OKB7 were
30
40
(#g/ml)
from C-ADE of HIV-1 infecmean of eight replicates of control represents mean of with HIV-l plus 1:20 human serum No. 200 (C’-ADE con35-0.5 pg/ml.
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TABLE 2 ANTI-HIV-~ ACTIVITIESOF MURINE MONOCLONALANTIBODIES Anti-HIV-l
Monoclonal
antibody
OKT4a OKT4f OKNK OKB7
Plus C’ADE’ kh-4
Minus C’-ADE* Wml)
0.06 NI” NI 1
0.20 NI NI NI
Percentage of epitope positive MT-2 cellsd
Anti-syncytiac Wml) 0.40 55 NI NI
96 81 0 90
BAnti-HIV-l plus C’-ADE titer was determined in the presence of 1:500 serum No. 200 and 1:20 human complement and refers to the concentration of antibody required to give >50% protection of cells from C’-ADE of HIV-1 infection. See Table 1. b Anti-HIV-l activity minus C’ADE was determined in the absence of complement as described in Table 1 and refers to the concentration of antibody required to give ~50% protection from HIV-l-induced cytopathic effect. c Anti-syncytia titer was determined as in Table 1 and refers to the concentration of antibody required to give >50% protection from HIV-1 induced syncytia formation after HIV-1 chronically infected H9 cells were mixed with uninfected MT-2 cells. d Flow cytometry using indirect immunofluorescence of unfixed MT-2 cells was performed on a Coulter EPICS 753 multiparameter flow cytometer. Control IgM and IgG were subtracted from values prior to calculation of percent positive cells. s No inhibition at any concentration tested. Highest concentration of all monoclonal antibodies tested was 50-60 pg/ml.
C’-ADE activity was confirmed using other parameters of HIV-l infection. As illustrated by Table 3, C’-ADE of HIV-l infection was blocked by both anti-CD4 and antiCR2 murine monoclonal antibodies when either HIV-l immunofluorescent (IF) positive cells or reverse transcriptase (RT) release was used as a marker of HIV-1 infection. When C’-ADE was assayed in the presence of 60 pg/ml of OKB7, only 50% of cells expressed HIV-
1 antigens compared to 100% of cells treated with enhancing antibody and OKNK or OKT4f at the same concentration. Cells challenged with virus and complement alone (no C’-ADE) were also 100% positive for HIV-1 antigen. OKT4a blocked C’ADE significantly as only 5% of cells were positive for HIV-1 antigens. When progeny virions were quantitated using RT release, OKB7 reduced the amount of RT from 2,105,OOOcpm/
TABLE 3 ANTI-HIV-~ ACTIVIN OF MURINE MONOCLONAL ANTBODIES CONFIRMEDBY PERCENTAGEANTIGEN POSITIVECELLS AND RT RELEASEFROM HIV-1 -CHALLENGED MT-2 CELLS Plus C’-ADEB
Monoclonal
antibody
OKNK OKT4a OKT4f HIV + Compl’ OKB7
Percentage IF positive cellsc looe 5 100 100 50
Minus C’-ADEb RT (cpm/ml)d
2,105,OOO 14,824 667,440 492,536 167,544
f 103,888 + 10,560 + 126,848 + 24,168 f 5,656
Percentage IF positive cells
RT (cpm/ml)
100 0 100 NTQ NT
2,748,773 + 169,268 13,453 + 9,840 1,494.960 + 100,277 NT NT
BAnti-HIV activity plus C’-ADE was determined in the presence of diluted (1:500) serum No. 200 and 1:20 human complement. Virus and antibodies were removed 24 hr after initial virus challenge. Results are for 72 hr after initial virus challenge. All monoclonal antibodies were tested at 60 pg/ml. * Anti-HIV activity minus C’-ADE was determined for the HTLV-Ills isolate of HIV-l. Virus and antibodies were removed 24 hr after initial virus challenge. Results are for 72 hr after the initial virus challenge. All monoclonal antibodies were tested at 60 pg/ml. c IF positive cells were quantitated using polyclonal human anti-HIV-l serum and FITC-conjugated goat anti-human IgG (Cappel) as described (34). d Reverse transcriptase release was quantitated using the method of Poiesz eta/. (3% ’ Denotes lysis of culture. ‘ HIV + 1:20 human complement and no enhancing antibody. Q Not tested.
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ml (OKNK) to 167,544 cpm/ml (Table 3). OKT4f had some anti-C’-ADE activity reducing RT release approximately 3-fold. OKT4a reduced RT release by greater than lOO-fold. HIV-l plus complement yielded 4-fold less RT release than the C’-ADE control. OKB7 had a greater effect on RT and HIV-1 antigen positive cells than the lack of enhancing antibody (complement plus virus alone) not because it had any anti-HIV activity but rather because dilute complement (1:40) alone has been shown to have weak HIV-1 infection-enhancing activity (16, 17). The anti-HIV-l activity of these monoclonal antibodies was similar whether tested in MT-2 cells in the absence of C’-ADE (Table 3) or in H9 cells (data not shown). Values of RT and IF positive cells cannot be compared for plus C’-ADE and minus C’-ADE in Table 3 because they are from two independent experiments. The data presented herein have shown that C’-ADE of HIV-1 infection in MT-2 cells requires complement receptors and CD4. The specific complement receptor required for C’-ADE of HIV-1 infection in MT-2 cells is CR2. It is possible that other complement receptors on other cells might also serve as receptors for C’-ADE of HIV-1 infection. CD4 was also required for both HIV1 infection and C’-ADE of HIV-1 infection as OKT4a, a potent anti-HIV-l monoclonal antibody (26-32), had both anti-HIV-1 and anti-C’-ADE activities. Although the exact role of complement and antibody to HIV-l in the natural pathogenesis of HIV-1 remains to be determined, it is apparent that cells bearing either Fc or complement receptors can be targets for HIV-l. The mechanism by which CR2 and complement enhance HIV-1 infection in vitro is unknown. However, HIV-1 opsonized with complement and anti-envelope antibodies may bind to cells with a higher affinity than nonopsonized virus binding to CD4 alone. Likewise, binding to CR2 may aid in virus penetration or uncoating or may cause alternative intracellular routing of HIV-l, thereby leading to increased replication efficiency as was hypothesized previously ( 17). Whatever the mechanism by which complement enhances infection in vitro, its in viva relevance is not understood. However, since the addition of complement can significantly reduce or completely abrogate the in vitro protective effects of HIV-l neutralizing antibodies present in heat-inactivated serum, it is imperative that the role of ADE in HIV1 infection in viva be determined. ACKNOWLEDGMENTS The authors thank Cecilia Haberzettl and William Covert of Ottho Diagnostics. Raritan, NJ, for the generous gift of murine monoclonal antibodies OKT4a. OKT4f, OKNK. and OKB7 used in these studies.
Critical discussions of the work were graciously provided by Drs. Tamar Ben-Porat. David Karzon. and Clark Tibbetts.
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