Protective Efficacy of Nonneutralizing Monoclonal Antibodies in Acute Infection with Murine Leukemia Virus

JOURNAL OF VIROLOGY, Nov. 1995, p. 7152 7158 Vol. 69, No. 11 0022-538X/95/$04.00 0 Copyright 1995, American Society for Microbiology Protective Efficacy of Nonneutralizing Monoclonal Antibodies in Acute Infection with Murine Leukemia Virus SETH H. PINCUS, ROBERT COLE, 1 * ROBIN IRELAND, 1 * FRANK MCATEE, 2 RYUICHI FUJISAWA, 2 AND JOHN PORTIS 2 Laboratory of Microbial Structure and Function 1 and Laboratory of Persistent Viral Diseases, 2 Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840 Received 29 March 1995/Accepted 14 August 1995 We have used an experimental retrovirus infection to study the roles played by different antibodies in resistance to both infection and disease. A molecularly cloned chimeric murine leukemia virus was used to induce acute lethal neurological disease in neonatal mice. A panel of monoclonal antibodies directed against the Gag and Env proteins was tested for protective efficacy. In vitro neutralization assays demonstrated that anti-env antibodies gave different degrees of neutralization, while no anti-gag neutralized the virus. In vivo experimental endpoints were onset of clinical signs and premoribund condition. As expected, different anti-env antibodies demonstrated different degrees of protection which correlated with their neutralizing abilities. Surprisingly, anti-gag antibodies directed against both p15 (MA protein) and p30 (CA protein) were also protective, significantly delaying the onset of disease. No protection was seen with either of two control antibodies. The protection with anti-gag was dose related and time dependent and was also produced with Fab fragments. Treatment with anti-gag did not prevent viremia but resulted in a slight slowing in viremia kinetics and decreased levels of virus in the central nervous systems of mice protected from disease. These data indicate that nonneutralizing antiretroviral antibodies can influence the outcome of retroviral disease. The data also suggest a functional role for cell surface expression of Gag proteins on murine leukemia virus-infected cells. Both passive and active antibody therapies are being considered for the prevention and/or treatment of human immunodeficiency virus (HIV) infection. It is generally accepted that the standard predictor of therapeutically effective antibody is in vitro neutralization of HIV infectivity, although the details of what exactly defines neutralizing antibody are not yet fully established (10, 12, 24 26, 32). Yet the majority of anti-hiv antibodies elicited by either infection or immunization are nonneutralizing (26). Some attention has been focused on nonneutralizing antibodies acting as potential enhancers of HIV infection by promoting Fc-mediated internalization of opsonized virions (1, 29). But any protective role played by such antibodies has largely been ignored. Because of the inherent difficulty of in vivo studies with HIV, we have used an animal model to study the effects of different antibodies in retroviral infection. Murine leukemia viruses (MuLV) can cause cytopathic disease in mice via multiple different mechanisms that have been defined at the molecular level (34). There are important differences between MuLV and HIV with regard to expression of viral antigens. One is the expression of a glycosylated Gag polyprotein (glyco-gag), a protein which is not incorporated into virions but is expressed at the surfaces of MuLV-infected cells (35). Although there have been reports of cell surface expression of HIV Gag proteins (14, 33) and anti-gag antibodies that neutralize HIV infectivity (21), most data do not support the idea that HIV Gag is exposed at the cell or virion surface. Almost 30 years ago, cell surface MuLV Gag was described as a tumor antigen in tumors associated with Gross virus. This structure was termed the Gross cell surface antigen * Corresponding author. Mailing address: Laboratory of Microbial Structure and Function, Rocky Mountain Laboratories, Hamilton, MT 59840. Phone: (406) 363-9317. Fax: (406) 363-9204. Present address: Department of Microbiology, Montana State University, Bozeman, MT 59715. (GCSA) and subsequently found to be glyco-gag. Antibody to the GCSA was able to protect against the lethal effects of transfer of syngeneic GCSA-positive leukemia cells (20). However, questions about the specificity of the protective antisera remain. A well-characterized panel of anti-mulv monoclonal antibodies has allowed us to readdress the issue of protective efficacy of anti-gag antibodies (2 4, 11, 17). The effects of passive transfer of antibody have been evaluated in an acute infection involving peripheral virus replication and central nervous system (CNS) disease manifesting itself within 2 to 3 weeks of infection (7, 27). We confirm that anti-gag antibodies are protective against disease manifestations, although they are less effective than neutralizing anti-env. Surprisingly, Fab fragments of anti-gag antibody also offered protection, suggesting a novel mechanism of action. MATERIALS AND METHODS Animals, antibodies, and viruses. IRW mice, of the Fv-1 n/n genotype and highly susceptible to neonatal MuLV infection (27, 34), were bred at the Rocky Mountain Laboratories. Experimental groups consisted of individual litters of neonatal mice, with 6 to 10 mice per litter. Animals were maintained with their mothers until they were 4 weeks of age. All of the monoclonal antibodies used in these studies have been described elsewhere (2 4, 11, 17, 22) and are summarized in Table 1. Antibodies were produced in tissue culture medium and purified on protein G-agarose (Zymed Laboratories, South San Francisco, Calif.). Fab fragments were made with immobilized papain (Pierce, Rockford, Ill.) and purified from Fc via protein A chromatography. The digestion and separation were confirmed by polyacrylamide gel electrophoresis and Western blotting (immunoblotting), (23) and antigen binding activity was confirmed by indirect immunofluorescence and flow cytometry (22). The chimeric MuLV designated FrCas E was created as a molecular clone by inserting env, derived from the neurovirulent wild-mouse ecotropic virus Cas BrE, into the genome of Friend MuLV clone FB29 (27). CasFr KP has the genome of CasBrE, with R-U5 and 5 leader sequences (bp 32 to 561) derived from FB29 (28). Stocks of infectious virus were prepared by transfecting viral DNA into Mus dunni cells. The virus stock was prepared in Fisher rat embryo cells. The titer of the virus stock was 1.1 10 6 focus-forming units per ml. All experiments were performed with the same virus stock, which was aliquoted into 7152

VOL. 69, 1995 ANTIBODY THERAPY FOR MURINE RETROVIRUS INFECTION 7153 TABLE 1. Monoclonal antibodies used in these studies Antibody Species and isotype a Specificity Neutralization b Reference 83 Rat, IgG2a MuLV gp70, polyreactive Weak 11 667 Mouse, IgG2a MuLV gp70, some ecotropic MuLV Yes 17 34 Mouse, IgG2b MuLV p15, polyreactive No 4 R187 Rat, IgG2 MuLV p30, polyreactive No 3 924 Mouse, IgG1 HIV gp120 No 22 DIII-A3 Mouse, IgG2b Chlamydia trachomatis MOMP c Not tested 36 a IgG, immunoglobulin G. b Summary of results from Fig. 1. Antibody 924 was tested in other experiments. c MOMP, major outer membrane protein. 1.0-ml volumes and frozen at 70 C. Murine L691b T cells were infected with FrCas E virus stocks and maintained in culture for more than 1 month before use. Infection of 95% of the cells was demonstrated by indirect immunofluorescence and flow cytometry (22) with both anti-gag and anti-env antibodies. Experimental infection. Mice were infected by intraperitoneal injection at 2 days of age with 5.5 10 2 focus-forming units in 50 l of saline. Purified antibody was administered intraperitoneally at a dose of 50 g unless otherwise noted. If antibody was administered on the same day as the virus, antibody and virus were premixed to avoid excess manipulation of the neonatal mice. Unless differently specified, antibody was administered three times: on the day of infection and 2 and 5 days postinfection. The kinetics of virus replication and details of the disease induced by FrCas E are highly reproducible and have been published elsewhere (7, 27). Mice developed neurologic signs, including motor disturbances and reflex abnormalities, 14 to 16 days postinfection, with progressive disease thereafter. Mice were euthanized when they reached a premoribund state. Experimental endpoints included onset of neurologic signs and time of euthanasia. In all experiments, these two endpoints paralleled each other. Results are presented as percent survival based on the number of mice surviving to 10 days of age. Deaths prior to 10 days were due to either neonatal mortality or the effects of manipulation and were not a result of the retrovirus infection. Statistical analysis was performed by using the Wilcoxon rank order test. In vitro assays of MuLV infectivity. Infectious virus was detected and quantified by a focal immunoassay (FIA) as described previously (6). Briefly, M. dunni fibroblast monolayers were incubated with the test virus in the presence of Polybrene (0.4 g/ml). After 3 days of additional culture, the monolayers were washed and immunoperoxidase stained with antibody 667. Viral foci were enumerated under a dissecting microscope. Antibody neutralization was performed by premixing the antibody and the virus, incubating the mixture for 1hat37 C, and transferring it to the cell monolayer. No complement was used. Determination of virus load in the CNS. Mice were infected at 1 day of age with the chimeric virus CasFr KP. This virus was chosen because it induces a slower onset of disease than does FrCas E (28); thus, disease status could be ascertained and animals could be sacrificed for analysis before they reached a moribund state. Mice were treated at 0, 2, and 5 days postinfection with either phosphate-buffered saline (PBS) or intact antibody R187 (50 g). Mice in each treatment group were evaluated for clinical signs at day 20 postinfection and then sacrificed. The brains were removed, washed, and then DNA extracted as described previously (27). DNA was digested with BamHI, which excises a 3.4-kb internal virus fragment. DNA (10 g) was loaded onto each lane of a 0.7% agarose gel, electrophoresed, and blotted onto nitrocellulose. Viral DNA was detected with a 32 P-labelled probe. The signal intensity at 3.4 kb was quantified with a Molecular Dynamics PhosphorImager. Results are given in arbitrary intensity units. with live cells and flow cytometry. As shown in Fig. 2, both anti-gag and anti-env recognize cell surface structures. The binding of the highly neutralizing antibody 667 was considerably greater than those of the other antibodies (28). In vivo protective efficacy of antibodies. Infection of mice within 2 days of birth with the chimeric MuLV FrCas E results in a peripheral viremia that peaks 4 days postinfection, enters the CNS, and reproducibly causes neurological signs commencing 14 to 17 days after the infection and death within another week (6, 8). If the peripheral viremia is delayed, either by lowering the inoculum or delaying the time of infection, then the onset of neurological disease is retarded and more variable (7, 27). The protective efficacies of two monoclonal antibodies directed against either Env or Gag, are shown in Fig. 3. Mice were infected with FrCas E at 2 days of age and received 50 g of antibody intraperitoneally 2 and 5 days postinfection. The onset of disease was significantly delayed (P 0.002) in both antibody groups compared with that in the PBS control group, but disease was not prevented. The results of a more detailed RESULTS Characterization of monoclonal antibodies. The abilities of monoclonal anti-mulv antibodies to neutralize in vitro infection by FrCas E were measured by FIA (Fig. 1). Anti-Env antibodies 83 and 667 both neutralized, although there were great differences in the neutralizing activity: complete neutralization was attained with antibody 667, while 83 gave only marginal inhibition at high concentrations. The mild degree of neutralization seen with 83 was consistent in several experiments. The magnitude of neutralization in these experiments corresponded with those in previously published reports describing these antibodies (11, 17). No neutralization was seen with either of the anti-gag antibodies, 34 and R187. Irrelevant control antibodies also failed to neutralize (data not shown). The binding of antibodies to FrCas E -infected L691b cells was determined by indirect immunofluorescence performed FIG. 1. Neutralization of FrCas E by monoclonal antibodies. A pretitered stock of cell-free virus at the appropriate dilution was mixed with the indicated concentration of antibody and incubated. The mixture was transferred to a monolayer of M. dunni cells; a standard FIA was then performed. The number of foci was counted and plotted against the antibody (Ab) concentration. Antibody 667 was used in the form of a hybridoma supernatant and is plotted with undiluted supernatant represented as the highest concentration.

7154 PINCUS ET AL. J. VIROL. FIG. 2. Indirect immunofluorescence with monoclonal antibodies. L691b T cells were persistently infected with FrCas E. Cells were stained with the indicated monoclonal antibody and then with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin antibody. The rat monoclonal antibodies cross-react extensively with this reagent. Three thousand cells were analyzed on a FACStar flow cytometer. Staining with the monoclonal antibody is shown in boldface; that of an irrelevant control antibody is shown in lightface. experiment are shown in Fig. 4. In this analysis, the negative controls were PBS and anti-hiv antibody 924, and virtually overlapping curves were obtained. Only results with 924 are shown. Antibodies to both Gag and Env were tested. Each antibody was administered twice, while another experimental group received three injections of anti-gag antibody 34 (at the time of infection and 2 and 5 days postinfection). Survival was significantly prolonged in all groups receiving anti-mulv antibodies, with no significant differences among the antibodies. FIG. 3. Protective efficacies of monoclonal antibodies. Three litters of IRW mice were infected at 2 days of age with FrCas E. At 2 and 5 days postinfection mice received 50 g of antibody intraperitoneally. The survival of mice within each experimental group is plotted against time. Survival in each antibody group was significantly extended compared with survival in the PBS control group (P 0.002 by Wilcoxon rank order test). DPI, days postinfection. FIG. 4. Multiple anti-mulv antibodies protect against experimental infection. Six litters of mice were infected 2 days after birth. All mice received 50 g of antibody 2 and 5 days postinfection. The group marked 3 doses received antibody at the time of infection as well. Negative controls, including an irrelevant antibody (924) and PBS (not shown), gave similar curves. Survival with all anti-mulv antibodies was significantly prolonged. The addition of a third dose of antibody improved survival over that of animals receiving two doses of the same antibody. DPI, days postinfection. The protection afforded by administering antibody three times was significantly better than that afforded by administering antibody twice. The experiment whose results are shown in Fig. 5 involved giving three injections of the different antibodies and included as a negative control the antichlamydial antibody DIII-A3, which is isotype matched to anti-gag antibody 34. Again, all antibodies significantly improved survival compared with the controls, and there was no distinction among the different antibodies. Results of a dose-response analysis of antibody R187 are shown in Fig. 6. The standard amount of 50 g was compared with 16.6 and 5 g per administration for three administrations. All gave statistically significant improvements in survival compared with no antibody. The greatest level of survival was seen at the highest dose of antibody, and this was statistically significantly different from the next lower dose. The protective effect of a highly efficient neutralizing antibody, anti-env 667, is shown in Fig. 7. This antibody completely prevented any manifestation of disease, probably by in vitro neutralization of the viral inoculum (which was mixed with the test antibody prior to injection). Additional experiments whose results are not shown here have demonstrated that anti-gag antibody R187 was also effective against a 10- fold-higher dose of FrCas E than was used in the experiments whose results are shown in Fig. 3 to 7. The addition of a fourth administration of R187 10 days postinfection did not have any additional protective effect. Effects of antibody therapy on viremia and CNS virus load. We have measured the level of viremia during the initial days postinfection in mice treated with antibody R187 and in control mice receiving no antibody. To obtain a measurable viremia within this time span, a 10-fold-higher inoculum of FrCas E (5.5 10 3 focus-forming units) was used. The results are

VOL. 69, 1995 ANTIBODY THERAPY FOR MURINE RETROVIRUS INFECTION 7155 FIG. 7. Neutralizing antibodies prevent disease. Mice were infected and given antibody as for Fig. 5. The neutralizing antibody 667 resulted in complete survival and also prevented the occurrence of symptoms. DPI, days postinfection. FIG. 5. Survival in animals receiving three doses of antibody. Mice were infected at 2 days of age and received 50 g of antibody 0, 2, and 5 days postinfection. DIII-A3 is isotype matched to antibody 34. Survival in all groups receiving anti-mulv was extended significantly compared with that in animals receiving DIII-A3 (P 0.01 to 0.002). DPI, days postinfection. shown in Fig. 8. A small decrease in viremia was seen in treated mice on days 3 to 5 postinfection, but following that time the difference between the groups disappeared. This experiment was performed with litters of four to seven mice for each time point. Table 2 shows the results and statistical analyses of two additional experiments. Beyond day 6, there were no differences between experimental and control groups. Thus, the in vivo administration of anti-gag antibodies delayed viremia but did not lower the peak level ultimately obtained. The period of time during which viremia was delayed has been shown to be a critical correlate of the extent of virus entry into the CNS (6 8, 27). Because animals were sacrificed prior to the development of disease, these data may minimize the effect of antibody treatment, since only a proportion of animals treated with antibody are protected from disease and protected animals may have lower levels of virus than those who develop disease. To determine whether antibody treatment resulted in a decreased virus load in the CNS, mice were infected with Cas- Fr KP, a related chimeric MuLV with slower disease kinetics (28). This allowed us to observe the development of disease FIG. 6. Dose response of antibody efficacy. Mice were infected at 2 days of age and received antibody 0, 2, and 5 days postinfection. Mice received 50, 16.6, or 5.5 g of R187 per administration. Survival was enhanced in all groups receiving antibody (P 0.02 to 0.002) compared with that in animals receiving PBS. Animals receiving 50 g of antibody survived longer than did those receiving the next lower dose (P 0.05). DPI, days postinfection. FIG. 8. Inhibition of early viremia by administration of anti-gag antibody. Litters of mice (four to seven mice per litter) were infected with virus at 2 days of age by using a 10-fold-larger inoculum than in all other experiments whose data are shown in other figures. Mice were treated with 50 g of R187 or PBS at days 0, 2, and 5 postinfection. Entire litters of mice were sacrificed and bled out on the indicated days. Virus titers in frozen serum samples were measured by FIA. Datum points are the geometric means.

7156 PINCUS ET AL. J. VIROL. TABLE 2. Viremia in treated and control mice Expt 1 Expt 2 Day Level of viremia in a : Statistical Level of viremia in: Control mice Treated mice significance b Control mice Treated mice Statistical significance 3 2.56 (0.16) 2.00 0.002 2.00 2.00 NT c 5 NT NT NT 3.72 (0.17) 1.85 (0.22) 0.0005 6 4.64 (0.02) 3.41 (0.46) 0.057 4.40 (0.19) 3.73 (0.15) 0.05 a Control mice received injections of PBS at days 0, 2, and 5 postinfection; treated mice received 50 g of R187 at each time point. Results are expressed as mean log focus-forming units per milliliter as determined by FIA; values in parentheses are standard errors of the means. b P by unpaired Student s t test. c NT, not tested. before animals were sacrificed for analysis. Mice were treated with either PBS or antibody R187. By 20 days postinfection, all mice in the PBS group developed neurological disease, while only 50% in the treatment group were ill. At this time animals were sacrificed, their brains were removed, and the virus load was determined by Southern blotting (Table 3). Animals protected from clinical disease by administration of antibody had significantly less viral DNA in the brain than control animals or treated animals who became ill (P 0.01). There was no difference between controls and those animals receiving antibody who developed clinical disease. Protection by Fab fragments. To understand the mechanism whereby nonneutralizing antibodies protect against the effects of infection, we made Fab fragments of R187. Binding of the Fab fragments to Gag was confirmed by indirect immunofluorescence. The absence of intact antibody in the Fab preparation was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting under both reducing and nonreducing conditions. FrCas E -infected mice were injected with equimolar quantities (based on number of antigen combining sites) of Fabs or intact antibodies. Fabs gave a significant level of protection compared with PBS, the negative control (Fig. 9). Although there appears to be greater protection with intact antibody than the Fab, this difference was not statistically significant in either experiment. Thus, at least some aspects of antibody-mediated protection were not induced by Fc-mediated effector mechanisms. The decreased protection with the Fabs compared with that with intact antibody may be due to the decreased avidity of Fabs compared with that of bivalent antibody, the result of some Fc-mediated effects playing a role in protection, or may not be of any real significance. In vitro effects of antibodies on FrCas E -infected cells. To study the mechanism whereby nonneutralizing antibodies may influence the development of viremia, we performed several in vitro analyses with L691b tissue culture cells persistently infected with FrCas E. More than 98% of these cells are infected and express both Gag and Env antigens (Fig. 2). To test whether antibody binding to the surfaces of infected cells can alter the amount of virus secreted, cells were incubated in the presence of nonneutralizing antibody and the amount of infectious virus released into the supernatant was measured by FIA (Fig. 10). None of the antibodies altered virus secretion. To see if antibody can block the cell-to-cell transmission of FrCas E, we plated infected L691b cells onto a monolayer of M. dunni cells in the presence or absence of antibody, and again the antibodies had no effect on infectivity (data not shown). We have also determined that antibody has no effect on proliferation and protein synthesis in infected L691b cells. Finally, we demonstrated that incubation of cells with anti-gag antibodies did not modulate the cell surface expression of Gag or Env. Although we failed to demonstrate any effect of anti-gag on these cultured T-cell tumor lines, we cannot rule out the pos- FIG. 9. Fab fragments have protective efficacy. Fab fragments were made from antibody R187. Mice were infected at 2 days of age and were treated with 50 g of intact antibody or 33 g of Fab at 0, 2, and 5 days postinfection. Results for two different experiments are shown. In both, the Fab fragment significantly improved survival compared with the PBS control (P 0.01 [A] and P 0.002 [B]). In neither experiment was the protection afforded by intact antibody significantly different from that obtained with Fab. DPI, days postinfection.

VOL. 69, 1995 ANTIBODY THERAPY FOR MURINE RETROVIRUS INFECTION 7157 TABLE 3. Virus load in CNSs of treated and control mice Treatment a CNS disease b No. of mice sibility that in vivo administration of the antibody may cause these effects in any of the various cells infected with FrCas E. DISCUSSION Viral DNA c PBS 8 4,085 732 0 NA d R187 6 4,215 1,353 6 2,374 259 e a Mice were injected with PBS or antibody R187 (50 g) on days 0, 2, and 5 postinfection. b Mice were scored for the presence or absence of CNS signs on day 20 postinfection. c Viral DNA was quantitated on a Southern blot with a Molecular Dynamics PhosphorImager. Values are arbitrary densitometric units and are the means the standard deviations of the values obtained for individual mice. d NA, not applicable. e P 0.01 by unpaired Student s t test compared with value for mice with CNS disease. The results in this paper demonstrate that nonneutralizing antibodies directed against viral core structures (Gag proteins CA and MA) can have a protective effect against disease induced by experimental retroviral infection, although the protective efficacy was less than that seen with neutralizing antibodies. The experimental model was acute neurological disease induced in neonatal mice by the chimeric murine leukemia virus FrCas E. The most likely target of these antibodies is glyco-gag. That protection was seen with Fab fragments of anti-gag antibody indicates that the mechanism of action was not simply the elimination of cells expressing glyco-gag by Fc-mediated mechanisms. Glyco-Gag contains a unique amino-terminal leader sequence that directs the unprocessed Gag polyprotein to the endoplasmic reticulum, where it is glycosylated and transferred to the cell membrane (9, 19, 31, 35). No glyco-gag is present FIG. 10. Addition of antibody to infected cells did not alter the rate of virus secretion. L691b cells (3 10 5 ), persistently infected with FrCas E, were incubated for 24 h in the presence or absence of 30 g of antibody in 1 ml. Supernatants were harvested, and the amount of infectious virus present was assayed by FIA. The number of foci counted is plotted against the dilution of supernatant tested. FFU, focus-forming units. on the surfaces of virions, and the function of glyco-gag is not known. Recent studies have shown that expression of glyco- Gag appears to facilitate the replication and/or spread of virus in vivo (5, 28). Mutant MuLV which lack expression of the protein exhibit slower replication during the first 1 to 2 weeks after inoculation than their respective wild-type viruses. For two mutants of a rapidly neurovirulent recombinant CasBrE virus, this alteration in replication kinetics was associated with a loss of pathogenicity within the CNS (28). Likewise, the capacity of Friend MuLV to cause acute hemolytic anemia was abrogated in a mutant lacking glyco-gag (5). It is therefore possible that the effect of the anti-gag antibodies on disease kinetics is a consequence of their capacity to inhibit or block the effect of glyco-gag on virus replication. The passively transferred antibody may act peripherally by altering the kinetics of viral replication, or it may act within the CNS. We favor the first hypothesis for three reasons. (i) Antibody reduced viremia during the period of peripheral replication (Fig. 8 and Table 2). (ii) Antibody decreased virus load in the CNSs of animals that were protected from disease. (iii) Antibody had the greatest effect when administered early after infection; administration of an extra dose of antibody at the time when neuropathology develops (10 days postinfection) had no additional salutary effect. Thus, by lowering the level of peripheral viremia during the first week postinfection, the amount of virus entering the CNS during this interval was decreased. This time period represents the developmental stage when virus best enters the CNS. Consequently, the development of neurological disease was delayed and in some cases prevented. This explanation is consistent with data demonstrating that other means that decrease the level of peripheral viremia during this time (viral alterations, a smaller inoculum, or delayed inoculation) inhibit the development of disease (7, 27, 28). The experiments reported here echo earlier studies with GCSA, which was later determined to be glyco-gag and which is found on the surfaces of leukemia cells induced with Gross MuLV (20). In those studies an antiserum reacting with GCSA offered complete protection against an otherwise-lethal challenge with GCSA-positive leukemia cells. The antiserum demonstrated in vitro complement-mediated cytotoxicity against GCSA-positive cells but not GCSA-negative cells. It is likely that the in vivo activity of this antiserum resulted from the removal of leukemia cells by complement and other Fc-mediated mechanisms. However, it should be noted that this antiserum reportedly had neutralizing activity against Gross virus. Since the antiserum was made by immunization with tumor cells, it is likely that it also contained anti-env. Thus, there is some difficulty in attributing all of the protective effect of anti-gcsa to anti-gag activity. Although this is the first report that nonneutralizing antibodies can inhibit retroviral infection in vivo, it is well established that nonneutralizing antibodies can protect against other viral infections, including vesicular stomatitis virus (15), Venezuelan equine encephalomyelitis virus (16), mouse hepatitis virus (18), Sindbis virus (30), and yellow fever virus (13). Some of these antibodies recognize structural proteins expressed on the surfaces of infected cells, while others recognize nonstructural proteins. In general, nonneutralizing antibodies were less efficacious or required higher concentrations than neutralizing antibodies. These studies indicate that anti-mulv Gag antibodies can play a role in inhibiting the pathogenesis of retroviral disease, probably by inhibiting virus replication. Though one may view MuLV infection as a model for human retroviral disease and find implications, the important differences between MuLV

7158 PINCUS ET AL. J. VIROL. and the human pathogenic retroviruses HIV and human T-cell leukemia virus type 1 should be kept in mind. Most efforts to develop both active and passive immunotherapies for human retroviral disease have focused on neutralizing anti-env antibodies, and our data confirm that neutralizing monoclonal antibodies have the greatest efficacy. Some attention has been focused on nonneutralizing antibodies as potentially enhancing HIV infection by promoting Fc-mediated internalization of opsonized virions (1, 29). But any protective role played by such antibodies has largely been ignored. Although the studies reported here focused on antibodies to MuLV glyco-gag, there is a direct analogy to nonneutralizing antibodies that bind to the HIV envelope protein. Both sets of determinants are expressed on infected cells, and antibodies may inhibit virus infection either by participating in the Fc-mediated elimination of infected cells or through a signalling mechanism. Such antibodies may be involved in protection against disease progression and may have a potential therapeutic role (22). ACKNOWLEDGMENTS We thank Susan and Carole Smaus for word processing assistance, Diane Brooks and Gerry Spangrude for assistance with flow cytometry, and John Swanson for manuscript review. This work supported by National Institute of Allergy and Infectious Diseases intramural research funds and by a biomedical research grant from the Katherine Dietz Award fund of the Arthritis Foundation. REFERENCES 1. Bolognesi, D. P. 1989. Do antibodies enhance the infection of cells by HIV? Nature (London) 340:431 432. 2. Britt, W. J., and B. Chesebro. 1983. Use of monoclonal anti-gp70 antibodies to mimic the effects of the Rfv-3 gene in mice with Friend virus-induced leukemia. J. Immunol. 130:2363 2367. 3. Chesebro, B., W. Britt, L. Evans, K. Wehrly, J. Nishio, and M. Cloyd. 1983. Characterization of monoclonal antibodies reactive with murine leukemia viruses: use in analysis of strains of Friend MCF and Friend ecotropic murine leukemia virus. Virology 127:134 148. 4. Chesebro, B., K. Wehrly, M. Cloyd, W. Britt, J. Portis, J. Collins, and J. Nishio. 1981. Characterization of mouse monoclonal antibodies specific for Friend murine leukemia virus-induced erythroleukemia cells: Friend-specific and FMR-specific antigens. Virology 112:131 144. 5. Corbin, A., A. C. Prats, J.-L. Darlix, and M. Sitbon. 1994. A nonstructural gag-encoded glycoprotein precursor is necessary for efficient spreading and pathogenesis of murine leukemia viruses. J. Virol. 68:3857 3867. 6. Czub, M., S. Czub, F. J. McAtee, and J. L. Portis. 1991. Age-dependent resistance to murine retrovirus-induced spongiform neurodegeneration results from central nervous system-specific restriction of virus replication. J. Virol. 65:2539 2544. 7. Czub, M., F. J. McAtee, and J. L. Portis. 1992. Murine retrovirus-induced spongiform encephalomyelopathy: host and viral factors which determine the length of the incubation period. J. Virol. 66:3298 3305. 8. Czub, S., W. P. Lynch, M. Czub, and J. L. Portis. 1994. Kinetic analysis of spongiform neurodegenerative disease induced by a highly virulent murine retrovirus. Lab. Invest. 70:711 723. 9. Edwards, S. A., and H. Fan. 1980. Sequence relationship of glycosylated and unglycosylated gag polyproteins of Moloney murine leukemia virus. J. Virol. 35:41 51. 10. Emini, E. A., W. A. Schleif, J. H. Nunberg, A. J. Conley, Y. Eda, S. Tokiyoshi, S. D. Putney, S. Matsushita, K. E. Cobb, C. M. Jett, J. W. Eichberg, and K. K. Murthy. 1992. Prevention of HIV-1 infection in chimpanzees by gp120 V3 domain-specific monoclonal antibody. Nature (London) 355:728 730. (Letter.) 11. Evans, L. H., R. P. Morrison, F. G. Malik, J. Portis, and W. J. Britt. 1990. A neutralizable epitope common to the envelope glycoproteins of ecotropic, polytropic, xenotropic, and amphotropic murine leukemia viruses. J. Virol. 64:6176 6183. 12. Golding, H., P. D Souza, J. Bradac, B. J. Mathieson, and P. Fast. 1994. The neutralization of HIV-1. AIDS Res. Hum. Retroviruses 10:633 643. 13. Gould, E. A., A. Buckley, A. D. T. Barrett, and N. Cammack. 1986. Neutralizing (54K) and non-neutralizing (54K and 48K) monoclonal antibodies against structural and non-structural yellow fever virus proteins confer immunity in mice. J. Gen. Virol. 67:591 595. 14. Laurent, A. G., B. Krust, M.-A. Rey, L. Montagnier, and A. G. Hovanessian. 1989. Cell surface expression of several species of human immunodeficiency virus type 1 major core protein. J. Virol. 63:4074 4078. 15. Lefrancois, L. 1984. Protection against lethal viral infection by neutralizing and nonneutralizing monoclonal antibodies: distinct mechanisms of action in vivo. J. Virol. 51:208 214. 16. Mathews, J. H., J. T. Roehrig, and D. W. Trent. 1985. Role of complement and the Fc portion of immunoglobulin G in immunity to Venezuelan equine encephalomyelitis virus infection with glycoprotein-specific monoclonal antibodies. J. Virol. 55:594 600. 17. McAtee, F. J., and J. L. Portis. 1985. Monoclonal antibodies specific for wild mouse neurotropic retrovirus: detection of comparable levels of virus replication in mouse strains susceptible and resistant to paralytic disease. J. Virol. 56:1018 1022. 18. Nakanaga, K., K. Yamanouchi, and K. Fujiwara. 1986. Protective effect of monoclonal antibodies on lethal mouse hepatitis virus infection in mice. J. Virol. 59:168 171. 19. Naso, R. B., L. H. Stanker, J. J. Kopchick, V. L. Ng, W. L. Karshin, and R. B. Arlinghaus. 1983. Further studies on the glycosylated gag gene products of Rauscher murine leukemia virus: identification of an N-terminal 45,000- dalton cleavage product. J. Virol. 45:1200 1206. 20. Old, L. J., E. Stockert, E. A. Boyse, and G. Geering. 1967. A study of passive immunization against a transplanted G leukemia with specific antiserum. Proc. Soc. Exp. Biol. Med. 124:63 68. 21. Papsidero, L. D., M. Sheu, and F. W. Ruscetti. 1989. Human immunodeficiency virus type 1-neutralizing monoclonal antibodies which react with p17 core protein: characterization and epitope mapping. J. Virol. 63:267 272. 22. Pincus, S. H., R. L. Cole, E. M. Hersh, D. Lake, Y. Masuho, P. J. Durda, and J. McClure. 1991. In vitro efficacy of anti-hiv immunotoxins targeted by various antibodies to the envelope protein. J. Immunol. 146:4315 4324. 23. Pincus, S. H., R. L. Cole, E. Kamanga-Sollo, and S. H. Fischer. 1993. Interaction of group B streptococcal opacity variants with the host defense system. Infect. Immun. 61:3761 3768. 24. Pincus, S. H., K. G. Messer, and S.-L. Hu. 1994. Effect of nonprotective vaccination on antibody response to subsequent human immunodeficiency virus infection. J. Clin. Invest. 93:140 146. 25. Pincus, S. H., K. G. Messer, P. L. Nara, W. A. Blattner, G. Colclough, and M. Reitz. 1994. Temporal analysis of the antibody response to HIV envelope protein in HIV-infected laboratory workers. J. Clin. Invest. 93:2505 2513. 26. Pincus, S. H., K. G. Messer, D. H. Schwartz, G. K. Lewis, B. S. Graham, W. A. Blattner, and G. Fisher. 1993. Differences in the antibody response to human immunodeficiency virus-1 envelope glycoprotein (gp160) in infected laboratory workers and vaccinees. J. Clin. Invest. 91:1987 1996. 27. Portis, J. L., S. Czub, C. F. Garon, and F. J. McAtee. 1990. Neurodegenerative disease induced by the wild mouse ecotropic retrovirus is markedly accelerated by long terminal repeat and gag-pol sequences from nondefective Friend murine leukemia virus. J. Virol. 64:1648 1656. 28. Portis, J. L., G. J. Spangrude, and F. J. McAtee. 1994. Identification of a sequence in the unique 5 open reading frame of the gene encoding glycosylated Gag which influences the incubation period of neurodegenerative disease induced by a murine retrovirus. J. Virol. 68:3879 3887. 29. Robinson, W. E., Jr., D. C. Montefiori, W. M. Mitchell, A. M. Prince, H. J. Alter, G. R. Dreesman, and J. W. Eichberg. 1989. Antibody-dependent enhancement of human immunodeficiency virus type 1 (HIV-1) infection in vitro by serum from HIV-1-infected and passively immunized chimpanzees. Proc. Natl. Acad. Sci. USA 86:4710 4714. 30. Schmaljohn, A. L., E. D. Johnson, J. M. Dalrymple, and G. A. Cole. 1982. Nonneutralizing monoclonal antibodies can prevent lethal alphavirus encephalitis. Nature (London) 297:70 72. 31. Schultz, A. M., E. H. Rabin, and S. Oroszlan. 1979. Post-translational modification of Rauscher leukemia virus precursor polyproteins encoded by the gag gene. J. Virol. 30:255 266. 32. Schwartz, D. H., G. Gorse, M. L. Clements, R. Belshe, A. Izu, A.-M. Duliege, P. Berman, T. Twadell, D. Stablein, R. Sposto, R. Siliciano, and T. Matthews. 1993. Induction of HIV-1-neutralising and syncytium-inhibiting antibodies in uninfected recipients of HIV-1 IIIB rgp120 subunit vaccine. Lancet 342:69 73. 33. Shang, F., H. Huang, K. Revesz, H.-C. Chen, R. Herz, and A. Pinter. 1991. Characterization of monoclonal antibodies against the human immunodeficiency virus matrix protein p17: identification of epitopes exposed at the surfaces of infected cells. J. Virol. 65:4798 4804. 34. Sitbon, M., B. Sola, L. Evans, J. Nishio, S. F. Hayes, K. Nathanson, C. F. Garon, and B. Chesebro. 1986. Hemolytic anemia and erythroleukemia, two distinct pathogenic effects of Friend MuLV. Mapping of the effects to different regions of the viral genome. Cell 47:851 859. 35. Tung, J. S., T. Yoshiki, and E. Fleissner. 1976. A core polyprotein of murine leukemia virus on the surface of mouse leukemia cells. Cell 9:573 578. 36. Zhang, Y.-X., S. J. Stewart, and H. D. Caldwell. 1989. Protective monoclonal antibodies to Chlamydia trachomatis serovar- and serogroup-specific major outer membrane protein determinants. Infect. Immun. 57:636 638.