JOURNAL OF VIROLOGY, Jan. 1990, p. 113-119
Vol. 64, No. 1
0022-538X/90/010113-07$02.00/0 Copyright X 1990, American Society for Microbiology
Antibody-Dependent Enhancement of Simian Immunodeficiency Virus (SIV) Infection In Vitro by Plasma from SIV-Infected Rhesus Macaques DAVID C. MONTEFIORI,1* W. EDWARD ROBINSON, JR.,' VANESSA M. HIRSCH,2 ANN MODLISZEWSKI,1 WILLIAM M. MITCHELL,1 AND PHILIP R. JOHNSON2 Department of Pathology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232,1 and Retroviral Pathogenesis Section, Division of Molecular Virology and Immunology, Department of Microbiology, Georgetown University, Rockville, Maryland 208522 Received 24 July 1989/Accepted 18 September 1989
Plasma from two rhesus macaques (Macaca mulaffa) experimentally infected with the simian immunodeficiency virus (SIV; isolate SIVmac251) enhanced SIVmac infection of a human CD4+ lymphoblastoid cell line, MT-2. Prechalienge plasma samples from these animals and serum from SIV-negative macaques did not enhance infection. Compared with controls, infection enhancement was characterized by the rapid appearance of syncytium formation (3 to 4 days sooner), reverse transcriptase release (10-fold increase), and cytopathic effect (60% cell killing). Enhancement of activity was dependent on the presence of diluted, fresh SIV-negative macaque serum as a source of complement. A requirement for complement was shown by the absence of enhancement in heat-inactivated serum and by dose-dependent inhibition of enhancement in the presence of polyclonal antibody to monkey complement component C3. Monoclonal antibody to CD4 (OKT4a) blocked enhancement completely, while monoclonal antibody to the human complement component C3d receptor CR2 (OKB7) reduced enhancement by greater than 50%, indicating a requirement for CD4 and CR2 in mediating this phenomenon. SIV infection-enhancing activity appeared in macaques soon after experimental inoculation (28 days). The titer increased over time and peaked just prior to the death of both macaques from opportunistic infections and lymphoma. In vitro SIV infection enhancement is nearly identical to the in vitro complementmediated, antibody-dependent enhancing (C'-ADE) activity observed in human immunodeficiency viruspositive human sera (Robinson et al., Lancet i:790-794, 1988; Robinson et al., J. Acq. Immun. Def. Synd. 2:33-42, 1989). These observations validate the macaque-SIV model for studies of C'-ADE.
Antibody-dependent enhancement (ADE) of viral infection is a phenomenon by which antibodies directed against a virus promote the infection process (26). The role of ADE in viral pathogenesis is uncertain, but it may contribute to the natural history of some viral diseases of animals and humans. ADE has been associated with enhanced viremias, shorter incubation periods from the time of infection to onset of disease, and increased disease severity (1, 9, 11, 22, 26, 28, 36, 40). For dengue virus infections, this association has been made by careful, prospective case studies in Southeast Asia, where dengue hemorrhagic fever and shock syndrome are endemic among children (18). For other viruses, this association has been made in vaccine studies with inactivated virus (9, 22, 26, 28, 36) or by passive immunization (1, 11, 40). ADE of viral infection in vitro occurs by at least two mechanisms. One mechanism operates through Fc receptors on macrophages which act as receptors for immunoglobulin G (IgG) or IgM complexed with virus. Once internalized via this mechanism, the virions are not effectively inactivated and can replicate within the macrophage. This type of ADE has been shown for dengue virus (10), West Nile virus (10), Sindbis virus (4), yellow fever virus (35), and, more recently, human immunodeficiency virus (HIV) (12, 37). Another mechanism for ADE in vitro involves immunoglobulin binding to the virus, followed by activation of the complement cascade. Complement components bound to the virus-antibody complex then interact with complement receptors on *
cells, and the complex is internalized. This mechanism of ADE has been shown for West Nile virus and dengue virus, in addition to the Fc-mediated ADE described above (3). Recently, complement-mediated ADE (C'-ADE) activity was observed in sera from HIV antibody-positive humans and chimpanzees, as well as passively immunized chimpanzees (29-33). C'-ADE of HIV infection in vitro increases infectious progeny virus production greater than 100-fold, with concomitant increases in reverse transcriptase (RT) release, viral antigen synthesis, and HIV RNA accumulation (32). This activity could be demonstrated in fresh human serum and Clq-deficient serum but not in serum treated with cobra venom anticomplementary protein or serum depleted of complement component C3 or factor B, indicating a requirement for the alternate pathway of complement (30, 33). HIV C'-ADE antibodies appear to belong to the IgG class, since they were present in high titer (1:65,000) (33) in a pooled HIV immunoglobulin fraction containing 97 to 98% IgG (27). Furthermore, antibodies conferring HIV C'-ADE are directed against the viral envelope glycoprotein (31). HIV C'-ADE activity has been found in over 85% of 85 HIV-positive individuals tested (32) and, when present, causes a dramatic reduction in the effective HIV neutralizing titer (30). The presence of infection-enhancing antibodies might have profound effects on the clinical outcome of HIVinduced disease. Furthermore, consideration of such antibody responses may be important elements in the design of safe and effective vaccine strategies. However, due to the lack of a convenient and appropriate animal model, the
Corresponding author. 113
114
MONTEFIORI ET AL.
significance of ADE in the natural course of HIV-induced disease has not been determined. Lack of an animal model has also hindered studies aimed at determining the effectiveness of HIV vaccines with ADE epitopes removed. Here we identify and characterize complement-mediated, simian immunodeficiency virus (SIV) infection-enhancing activity in the plasma of SIV-infected rhesus macaques and find that it closely resembles the C'-ADE activity observed in HIVpositive human sera. MATERIALS AND METHODS
Cells and virus. The human T-lymphotropic virus type I (HTLV-I)-transformed, CD4+ CR2+ lymphoblastoid cell line MT-2 was a gift from D. Richman, the University of California-San Diego Veterans Administration Medical Center. H9 cells were obtained from R. C. Gallo, National Cancer Institute. SIVmac251 (originally provided by N. Letvin, New England Regional Primate Center) was prepared from conditioned culture fluids of chronically infected H9 cells. Virus stocks were made cell-free by low-speed centrifugation and filtration (0.45 ptm filters) and used immediately. Virus was quantitated by RT activity, measured as described before (25). All cultures were maintained in RPMI1640 medium containing 12% heat-inactivated fetal bovine serum and 50 jig of gentamicin per ml. Macaque infections and blood products. Heparinized plasma samples were collected from two rhesus macaques (nos. 648 and 662) 7 days before inoculation and 28, 122, and 270 days after inoculation with undiluted virus stock. The SIV stock used to infect the macaques was obtained from culture supernatants of H9 cells chronically infected with SIVmac251; supernatants were prepared as described above and stored in liquid nitrogen. Antibodies to all major SIV antigens were detected in both macaques by Western immunoblot at 28 days after inoculation. SIV was recovered by cocultivation of peripheral blood mononuclear cells with H9 cells at monthly intervals until death occurred. Both animals manifested immunodeficiency (low absolute CD4 count) within 1 month post-inoculation. Animal 662 died on day 280 with lymphoma and pneumonia. Animal 648 died on day 331. Fresh-frozen control serum obtained from healthy, SIVnegative (as determined by immunofluorescence and enzyme immunoassay) rhesus macaques was used as a source of complement. Polyclonal and monoclonal antibodies. Goat anti-monkey C3 polyclonal antiserum was obtained from Organon TeknikalCappel Laboratories, West Chester, Pa. OKT4a (anti-CD4) and OKB7 (anti-CR2) monoclonal antibodies were a generous gift from Cecilia Haberzettl and William Covert, Ortho Diagnostic Systems, Raritan, N.J. Measurement of enhanced SIV infection. SIV infection enhancement was measured by using an MT-2 cell microtiter infection assay similar to that described for HIV (23, 30). All plasma samples were heat-inactivated at 56°C for 1 h prior to assay. Enhancing titers were determined by performing twoor threefold serial dilutions of macaque plasma samples in triplicate in 96-well microdilution plates. The diluent was growth medium containing 1:20-diluted macaque complement-containing serum. Virus was added and incubated for 1 h at 37°C (final dilution of complement on virus was 1:40). MT-2 cells were then added, and the plates were incubated until syncytium formation and cytopathic effect were observed microscopically. The plates were then harvested for measurement of viable cells in each well by a vital dye uptake method (23). In some cases, the remaining culture
J. VIROL.
volumes at each dilution were pooled and assayed for RT activity. The range for quantitating percent viable cells was determined from the difference in A540 between the average of eight nonenhancing control wells (cells plus virus plus complement but no enhancing plasma) and the average of eight blank wells containing no cells or virus. Vital dye uptake in this assay is linear from A540 readings of 0.025 to 0.85, which correspond to 2 x 104 to 25 x 104 viable cells per well (23). In experiments designed to examine the ability of specific antibodies to block SIV infection enhancement, the antibodies were twofold serially diluted in triplicate in 96-well microdilution plates containing growth medium and a constant amount of macaque complement and enhancing plasma as indicated. The range for calculating percent viable cells in these experiments was determined by the difference in A540 between the average of eight nonenhancing wells (cells plus virus plus complement but no enhancing plasma) and the average of eight wells with full enhancing activity (cells plus virus plus complement plus enhancing plasma). Standard deviations for both assays were less than 10% for each data point. RESULTS SIV infection-enhancing activity. Infection-enhancing activity in SIV-positive macaque plasma was measured on MT-2 cells by syncytium formation, cytopathic effect, and release of RT activity into the culture fluids. Each of these measures of infection occurred much sooner in the presence of infection-enhancing plasma than in its absence. An example of the rapid appearance of SIV-induced syncytium formation caused by SIV-positive macaque plasma is shown in Fig. 1. Here, 8 days after virus was added, about half of the cells challenged in the presence of infection-enhancing plasma were involved in syncytia (right panel). At the same time, no syncytia were observed when cells were challenged in the presence of complement but lacking enhancing plasma (left panel). Another 3 to 4 days were required before the nonenhanced infections became involved in syncytium formation to the same extent as the cells in the right panel. The rapid appearance of syncytium formation induced by plasma from macaque 648 was -accompanied by increased cytopathic effect (over 60% killing of cells) and dramatic rises in RT activity (up to 10-fold increases) found in culture supernatants (Fig. 2). These effects were seen at a time when no syncytia or cytopathic effects were observed in nonenhanced control infections. Plasma from this animal and from macaque 662 had enhancing activity when collected 28, 120, and 270 days postinoculation (Fig. 2 and Table- 1). Enhancing titers were relatively low at 28 days and increased to peak levels at 122 and 270 days (Table 1). Some neutralizing activity was present at low dilutions (1:18 to 1:72) of macaque 648 plasma samples from days 122 and 270; cytopathic effects and rises in RT activities were less pronounced. Strongest enhancement was not observed until a dilution of 1:288 was reached (Fig. 2). Plasma from macaque 662, on the other hand, had neutralizing activity only at a 1:18 dilution for the day 122 sample (data not shown). No enhancing activity was present in preinfection plasma from these animals, nor was it present in serum from SIV-negative macaques. Role of complement. SIV infection enhancement required fresh, SIV-negative macaque serum. This was very similar to C'-ADE of HIV infection, which requires fresh, HIVnegative serum as a source of alternate complement pathway
C'-ADE OF SIV INFECTION
VOL. 64, 1990 ...
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FIG. 1. Effect of SIV infection-enhancing plasma on syncytium formation in MT-2 cells. MT-2 cells were challenged with SIVmac251 in the presence of a 1:40 dilution of macaque complement-containing serum without (left) and with (right) a 1:288 dilution of plasma from macaque 648 (day 270). Photographs were taken 8 days post-virus challenge.
activity (30). Heat-inactivated (a process used to destroy complement) SIV-positive plasma and fresh SIV-negative serum alone had no enhancing activity. However, in the presence of 1:40 SIV-negative serum, strong enhancing activity of SIV-positive plasma was observed (Fig. 1 and 2, Table 1). Enhancement was lost when SIV-positive plasma and SIV-negative serum were both heat-inactivated (data not shown). A requirement for complement in C'-ADE of HIV infection with human serum suggested a similar requirement in ADE of SIV infection. The loss of SIV infection-enhancing activity by heat inactivation supported this requirement. Therefore, we investigated the ability of anti-monkey complement component C3 antibody to block SIV infection enhancement. This antibody suppressed the SIV infectionenhancing activity of macaque 648 plasma (day 270) in a dose-dependent manner. Ninety percent of the activity was suppressed at a dilution of 1:16 (Fig. 3), confirming the complement requirement for SIV infection enhancement. The titer and strength of SIV infection-enhancing activity might be limited by species variation in the complement system. Specifically, monkey complement components may not interact with human complement receptors as well as they do with monkey complement receptors. Since human cells were being used to measure C'-ADE of SIV infection, fresh human serum was substituted for fresh macaque serum in some assays. SIV infection enhancement by macaque 662 plasma (day 270) was greater in fresh human serum than in
(Fig. 4). Whereas infection enhanceserum to a dilution of 1:112, infection enhancement was observed with human serum to a dilution of 1:448. In addition, the extent of cytopathic effect observed was up to 43% greater in human serum than in macaque serum (95 versus 52% viable cells for macaque versus human serum at a dilution of 1:112). Receptors for SIV infection enhancement. The requirement for complement in SIV infection enhancement suggested a role for complement receptors. MT-2 cells have been shown by immunofluorescence assay and flow cytometry to express CR2 but not CR1 or CR3 (32). Therefore, a monoclonal antibody against CR2 (OKB7) was tested for the ability to block C'-ADE of SIV infection. Also, since CD4 acts as receptor for both HIV and SIV under nonenhanced conditions, a monoclonal antibody to CD4 (OKT4a) which blocks nonenhanced HIV infections (21) was tested to determine the CD4 requirement in SIV infection enhancement. A monoclonal antibody to CD4 (OKT4c) which does not efficiently block HIV infection (21) was used as a negative control. The OKT4a monoclonal antibody at 1.25 ,ug/ml blocked greater than 90% of SIV infection-enhancing activity found in macaque 648 plasma (day 270) (Fig. 5). Lower concentrations of OKT4a monoclonal antibody elicited dose-dependent inhibition; at the lowest concentration tested (0.16 ,ug/ml), it resulted in 45% inhibition. OKB7 monoclonal antibody reduced infection enhancement by
fresh
macaque serum
ment was observed in macaque
J. VIROL.
MONTEFIORI ET AL.
116
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FIG. 3. Reduction of SIVmac251 infection enhancement in MT-2 cells in the presence of anti-monkey complement component C3 antibody. Anti-monkey C3 antiserum was tested aiti dilutions of 1:16 to 1:2,048 for the ability to block the SIV infection-enhancing activity of a 1:750 dilution of macaque 648 plasma (day 270). Macaque complement-containing serum was present at a 1:40 dilution. The maximum range in A540 for calculating percent reduction in C'-ADE was 0.23 unit, corresponding to 33% reduction in viable cells.
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Vol. 64, No. 1
0022-538X/90/010113-07$02.00/0 Copyright X 1990, American Society for Microbiology
Antibody-Dependent Enhancement of Simian Immunodeficiency Virus (SIV) Infection In Vitro by Plasma from SIV-Infected Rhesus Macaques DAVID C. MONTEFIORI,1* W. EDWARD ROBINSON, JR.,' VANESSA M. HIRSCH,2 ANN MODLISZEWSKI,1 WILLIAM M. MITCHELL,1 AND PHILIP R. JOHNSON2 Department of Pathology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232,1 and Retroviral Pathogenesis Section, Division of Molecular Virology and Immunology, Department of Microbiology, Georgetown University, Rockville, Maryland 208522 Received 24 July 1989/Accepted 18 September 1989
Plasma from two rhesus macaques (Macaca mulaffa) experimentally infected with the simian immunodeficiency virus (SIV; isolate SIVmac251) enhanced SIVmac infection of a human CD4+ lymphoblastoid cell line, MT-2. Prechalienge plasma samples from these animals and serum from SIV-negative macaques did not enhance infection. Compared with controls, infection enhancement was characterized by the rapid appearance of syncytium formation (3 to 4 days sooner), reverse transcriptase release (10-fold increase), and cytopathic effect (60% cell killing). Enhancement of activity was dependent on the presence of diluted, fresh SIV-negative macaque serum as a source of complement. A requirement for complement was shown by the absence of enhancement in heat-inactivated serum and by dose-dependent inhibition of enhancement in the presence of polyclonal antibody to monkey complement component C3. Monoclonal antibody to CD4 (OKT4a) blocked enhancement completely, while monoclonal antibody to the human complement component C3d receptor CR2 (OKB7) reduced enhancement by greater than 50%, indicating a requirement for CD4 and CR2 in mediating this phenomenon. SIV infection-enhancing activity appeared in macaques soon after experimental inoculation (28 days). The titer increased over time and peaked just prior to the death of both macaques from opportunistic infections and lymphoma. In vitro SIV infection enhancement is nearly identical to the in vitro complementmediated, antibody-dependent enhancing (C'-ADE) activity observed in human immunodeficiency viruspositive human sera (Robinson et al., Lancet i:790-794, 1988; Robinson et al., J. Acq. Immun. Def. Synd. 2:33-42, 1989). These observations validate the macaque-SIV model for studies of C'-ADE.
Antibody-dependent enhancement (ADE) of viral infection is a phenomenon by which antibodies directed against a virus promote the infection process (26). The role of ADE in viral pathogenesis is uncertain, but it may contribute to the natural history of some viral diseases of animals and humans. ADE has been associated with enhanced viremias, shorter incubation periods from the time of infection to onset of disease, and increased disease severity (1, 9, 11, 22, 26, 28, 36, 40). For dengue virus infections, this association has been made by careful, prospective case studies in Southeast Asia, where dengue hemorrhagic fever and shock syndrome are endemic among children (18). For other viruses, this association has been made in vaccine studies with inactivated virus (9, 22, 26, 28, 36) or by passive immunization (1, 11, 40). ADE of viral infection in vitro occurs by at least two mechanisms. One mechanism operates through Fc receptors on macrophages which act as receptors for immunoglobulin G (IgG) or IgM complexed with virus. Once internalized via this mechanism, the virions are not effectively inactivated and can replicate within the macrophage. This type of ADE has been shown for dengue virus (10), West Nile virus (10), Sindbis virus (4), yellow fever virus (35), and, more recently, human immunodeficiency virus (HIV) (12, 37). Another mechanism for ADE in vitro involves immunoglobulin binding to the virus, followed by activation of the complement cascade. Complement components bound to the virus-antibody complex then interact with complement receptors on *
cells, and the complex is internalized. This mechanism of ADE has been shown for West Nile virus and dengue virus, in addition to the Fc-mediated ADE described above (3). Recently, complement-mediated ADE (C'-ADE) activity was observed in sera from HIV antibody-positive humans and chimpanzees, as well as passively immunized chimpanzees (29-33). C'-ADE of HIV infection in vitro increases infectious progeny virus production greater than 100-fold, with concomitant increases in reverse transcriptase (RT) release, viral antigen synthesis, and HIV RNA accumulation (32). This activity could be demonstrated in fresh human serum and Clq-deficient serum but not in serum treated with cobra venom anticomplementary protein or serum depleted of complement component C3 or factor B, indicating a requirement for the alternate pathway of complement (30, 33). HIV C'-ADE antibodies appear to belong to the IgG class, since they were present in high titer (1:65,000) (33) in a pooled HIV immunoglobulin fraction containing 97 to 98% IgG (27). Furthermore, antibodies conferring HIV C'-ADE are directed against the viral envelope glycoprotein (31). HIV C'-ADE activity has been found in over 85% of 85 HIV-positive individuals tested (32) and, when present, causes a dramatic reduction in the effective HIV neutralizing titer (30). The presence of infection-enhancing antibodies might have profound effects on the clinical outcome of HIVinduced disease. Furthermore, consideration of such antibody responses may be important elements in the design of safe and effective vaccine strategies. However, due to the lack of a convenient and appropriate animal model, the
Corresponding author. 113
114
MONTEFIORI ET AL.
significance of ADE in the natural course of HIV-induced disease has not been determined. Lack of an animal model has also hindered studies aimed at determining the effectiveness of HIV vaccines with ADE epitopes removed. Here we identify and characterize complement-mediated, simian immunodeficiency virus (SIV) infection-enhancing activity in the plasma of SIV-infected rhesus macaques and find that it closely resembles the C'-ADE activity observed in HIVpositive human sera. MATERIALS AND METHODS
Cells and virus. The human T-lymphotropic virus type I (HTLV-I)-transformed, CD4+ CR2+ lymphoblastoid cell line MT-2 was a gift from D. Richman, the University of California-San Diego Veterans Administration Medical Center. H9 cells were obtained from R. C. Gallo, National Cancer Institute. SIVmac251 (originally provided by N. Letvin, New England Regional Primate Center) was prepared from conditioned culture fluids of chronically infected H9 cells. Virus stocks were made cell-free by low-speed centrifugation and filtration (0.45 ptm filters) and used immediately. Virus was quantitated by RT activity, measured as described before (25). All cultures were maintained in RPMI1640 medium containing 12% heat-inactivated fetal bovine serum and 50 jig of gentamicin per ml. Macaque infections and blood products. Heparinized plasma samples were collected from two rhesus macaques (nos. 648 and 662) 7 days before inoculation and 28, 122, and 270 days after inoculation with undiluted virus stock. The SIV stock used to infect the macaques was obtained from culture supernatants of H9 cells chronically infected with SIVmac251; supernatants were prepared as described above and stored in liquid nitrogen. Antibodies to all major SIV antigens were detected in both macaques by Western immunoblot at 28 days after inoculation. SIV was recovered by cocultivation of peripheral blood mononuclear cells with H9 cells at monthly intervals until death occurred. Both animals manifested immunodeficiency (low absolute CD4 count) within 1 month post-inoculation. Animal 662 died on day 280 with lymphoma and pneumonia. Animal 648 died on day 331. Fresh-frozen control serum obtained from healthy, SIVnegative (as determined by immunofluorescence and enzyme immunoassay) rhesus macaques was used as a source of complement. Polyclonal and monoclonal antibodies. Goat anti-monkey C3 polyclonal antiserum was obtained from Organon TeknikalCappel Laboratories, West Chester, Pa. OKT4a (anti-CD4) and OKB7 (anti-CR2) monoclonal antibodies were a generous gift from Cecilia Haberzettl and William Covert, Ortho Diagnostic Systems, Raritan, N.J. Measurement of enhanced SIV infection. SIV infection enhancement was measured by using an MT-2 cell microtiter infection assay similar to that described for HIV (23, 30). All plasma samples were heat-inactivated at 56°C for 1 h prior to assay. Enhancing titers were determined by performing twoor threefold serial dilutions of macaque plasma samples in triplicate in 96-well microdilution plates. The diluent was growth medium containing 1:20-diluted macaque complement-containing serum. Virus was added and incubated for 1 h at 37°C (final dilution of complement on virus was 1:40). MT-2 cells were then added, and the plates were incubated until syncytium formation and cytopathic effect were observed microscopically. The plates were then harvested for measurement of viable cells in each well by a vital dye uptake method (23). In some cases, the remaining culture
J. VIROL.
volumes at each dilution were pooled and assayed for RT activity. The range for quantitating percent viable cells was determined from the difference in A540 between the average of eight nonenhancing control wells (cells plus virus plus complement but no enhancing plasma) and the average of eight blank wells containing no cells or virus. Vital dye uptake in this assay is linear from A540 readings of 0.025 to 0.85, which correspond to 2 x 104 to 25 x 104 viable cells per well (23). In experiments designed to examine the ability of specific antibodies to block SIV infection enhancement, the antibodies were twofold serially diluted in triplicate in 96-well microdilution plates containing growth medium and a constant amount of macaque complement and enhancing plasma as indicated. The range for calculating percent viable cells in these experiments was determined by the difference in A540 between the average of eight nonenhancing wells (cells plus virus plus complement but no enhancing plasma) and the average of eight wells with full enhancing activity (cells plus virus plus complement plus enhancing plasma). Standard deviations for both assays were less than 10% for each data point. RESULTS SIV infection-enhancing activity. Infection-enhancing activity in SIV-positive macaque plasma was measured on MT-2 cells by syncytium formation, cytopathic effect, and release of RT activity into the culture fluids. Each of these measures of infection occurred much sooner in the presence of infection-enhancing plasma than in its absence. An example of the rapid appearance of SIV-induced syncytium formation caused by SIV-positive macaque plasma is shown in Fig. 1. Here, 8 days after virus was added, about half of the cells challenged in the presence of infection-enhancing plasma were involved in syncytia (right panel). At the same time, no syncytia were observed when cells were challenged in the presence of complement but lacking enhancing plasma (left panel). Another 3 to 4 days were required before the nonenhanced infections became involved in syncytium formation to the same extent as the cells in the right panel. The rapid appearance of syncytium formation induced by plasma from macaque 648 was -accompanied by increased cytopathic effect (over 60% killing of cells) and dramatic rises in RT activity (up to 10-fold increases) found in culture supernatants (Fig. 2). These effects were seen at a time when no syncytia or cytopathic effects were observed in nonenhanced control infections. Plasma from this animal and from macaque 662 had enhancing activity when collected 28, 120, and 270 days postinoculation (Fig. 2 and Table- 1). Enhancing titers were relatively low at 28 days and increased to peak levels at 122 and 270 days (Table 1). Some neutralizing activity was present at low dilutions (1:18 to 1:72) of macaque 648 plasma samples from days 122 and 270; cytopathic effects and rises in RT activities were less pronounced. Strongest enhancement was not observed until a dilution of 1:288 was reached (Fig. 2). Plasma from macaque 662, on the other hand, had neutralizing activity only at a 1:18 dilution for the day 122 sample (data not shown). No enhancing activity was present in preinfection plasma from these animals, nor was it present in serum from SIV-negative macaques. Role of complement. SIV infection enhancement required fresh, SIV-negative macaque serum. This was very similar to C'-ADE of HIV infection, which requires fresh, HIVnegative serum as a source of alternate complement pathway
C'-ADE OF SIV INFECTION
VOL. 64, 1990 ...
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FIG. 1. Effect of SIV infection-enhancing plasma on syncytium formation in MT-2 cells. MT-2 cells were challenged with SIVmac251 in the presence of a 1:40 dilution of macaque complement-containing serum without (left) and with (right) a 1:288 dilution of plasma from macaque 648 (day 270). Photographs were taken 8 days post-virus challenge.
activity (30). Heat-inactivated (a process used to destroy complement) SIV-positive plasma and fresh SIV-negative serum alone had no enhancing activity. However, in the presence of 1:40 SIV-negative serum, strong enhancing activity of SIV-positive plasma was observed (Fig. 1 and 2, Table 1). Enhancement was lost when SIV-positive plasma and SIV-negative serum were both heat-inactivated (data not shown). A requirement for complement in C'-ADE of HIV infection with human serum suggested a similar requirement in ADE of SIV infection. The loss of SIV infection-enhancing activity by heat inactivation supported this requirement. Therefore, we investigated the ability of anti-monkey complement component C3 antibody to block SIV infection enhancement. This antibody suppressed the SIV infectionenhancing activity of macaque 648 plasma (day 270) in a dose-dependent manner. Ninety percent of the activity was suppressed at a dilution of 1:16 (Fig. 3), confirming the complement requirement for SIV infection enhancement. The titer and strength of SIV infection-enhancing activity might be limited by species variation in the complement system. Specifically, monkey complement components may not interact with human complement receptors as well as they do with monkey complement receptors. Since human cells were being used to measure C'-ADE of SIV infection, fresh human serum was substituted for fresh macaque serum in some assays. SIV infection enhancement by macaque 662 plasma (day 270) was greater in fresh human serum than in
(Fig. 4). Whereas infection enhanceserum to a dilution of 1:112, infection enhancement was observed with human serum to a dilution of 1:448. In addition, the extent of cytopathic effect observed was up to 43% greater in human serum than in macaque serum (95 versus 52% viable cells for macaque versus human serum at a dilution of 1:112). Receptors for SIV infection enhancement. The requirement for complement in SIV infection enhancement suggested a role for complement receptors. MT-2 cells have been shown by immunofluorescence assay and flow cytometry to express CR2 but not CR1 or CR3 (32). Therefore, a monoclonal antibody against CR2 (OKB7) was tested for the ability to block C'-ADE of SIV infection. Also, since CD4 acts as receptor for both HIV and SIV under nonenhanced conditions, a monoclonal antibody to CD4 (OKT4a) which blocks nonenhanced HIV infections (21) was tested to determine the CD4 requirement in SIV infection enhancement. A monoclonal antibody to CD4 (OKT4c) which does not efficiently block HIV infection (21) was used as a negative control. The OKT4a monoclonal antibody at 1.25 ,ug/ml blocked greater than 90% of SIV infection-enhancing activity found in macaque 648 plasma (day 270) (Fig. 5). Lower concentrations of OKT4a monoclonal antibody elicited dose-dependent inhibition; at the lowest concentration tested (0.16 ,ug/ml), it resulted in 45% inhibition. OKB7 monoclonal antibody reduced infection enhancement by
fresh
macaque serum
ment was observed in macaque
J. VIROL.
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@ 28 d * 122 d * 270 d
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FIG. 3. Reduction of SIVmac251 infection enhancement in MT-2 cells in the presence of anti-monkey complement component C3 antibody. Anti-monkey C3 antiserum was tested aiti dilutions of 1:16 to 1:2,048 for the ability to block the SIV infection-enhancing activity of a 1:750 dilution of macaque 648 plasma (day 270). Macaque complement-containing serum was present at a 1:40 dilution. The maximum range in A540 for calculating percent reduction in C'-ADE was 0.23 unit, corresponding to 33% reduction in viable cells.
100 90 x E
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50
p
40 30 20 10 0
0
