Proc. Natl. Acad. Sci. USA

Vol. 74, No. 6, pp. 2546-2550, June 1977 Immunology

Isolation of virus-free Herpesvirus saimiri antigen-positive plasma membrane vesicles (vaccine/immunity/primates/neutralizing antibody)

GARY R. PEARSON* AND ROBERT E. SCOTTt Departments of Microbiology*, and Pathology and Anatomyt, Mayo Clinic/Foundation, Rochester, Minnesota 55901

Communicated by George Klein, April,4, 1977

ABSTRACT Experiments were undertaken to determine whether virus-free, antigen-positive plasma membrane vesicles could be isolated from owl monkeykidney cells infected with Herpesvirus saimiri. The results demonstrate that vesicles can be produced by using a vesiculation fluid containing 25 mM formaldehyde/2 mM dithiothreitol. Electron microscopy revealed that these vesicles were free of detectable virus particles. Vesicles prepared from the infected cells contained virus-induced membrane antigens as shown by membrane immunofluorescence and by inhibition of antibody-dependent lymphocyte cytotoxicity. Nonhuman primates immunized with vesicles produced antibodies to these membrane antigens, late cytoplasmic antigens, and neutralizing antibodies. Infectious virus was not demonstrated in these vesicles by cocultivation with owl monkey kidney cells or by the inoculation of cottontop marmosets. Furthermore, no DNA could be demonstrated in vesicles prepared from Herpesvirus-infected owl monkey kidney cells. The implication of these findings in relation to the question of a virus-free membrane vaccine against Herpesvirus infections is discussed.

Herpesviruses have received considerable attention in the past few years largely because of their association with animal and human cancers. A number of herpesviruses have now been demonstrated to induce cancers in animal systems (1-6). Numerous investigations have also linked two human herpesviruses with some human malignancies: the Epstein-Barr virus, implicated in the etiology of African Burkitt's lymphoma and nasopharyngeal carcinoma (7), and herpes simplex virus type 2, associated with cervical cancer (8-10). These findings raise the possibility that prevention of these cancers might be feasible through the use of viral vaccines. In support of this proposal, it has been demonstrated that Marek's disease lymphoma in chickens can be prevented by immunization with a live avirulent herpes virus vaccine (11, 12). Furthermore, Laufs and Steinke (13) reported that Herpesvirus saimiri (HVS)-induced lymphoma in cottontop marmosets was prevented by immunization with inactivated HVS. These findings demonstrate that it is indeed possible to prevent the induction of herpes virus-induced malignancies with appropriate viral vaccines. However, because of the potential danger of using attehuated or inactivated viral vaccines containing viral genetic information for the immunization of human populations, as recently discussed by Epstein (14), it would be more realistic to use purified herpesvirus protein, free of virus and viral DNA, as a vaccine for the induction of immunity in susceptible populations. In most of the herpesvirus systems studied so far, virusspecified antigens have been detected in the plasma membrane of virus-infected or transformed cells (15-20). Investigations

on membrane antigens (MA) induced by HSV, Epstein-Barr virus, and Marek's disease herpesvirus have provided evidence that these antigens are also expressed in the envelope of infectious virus particles and are probably responsible for the induction of neutralizing antibodies against the specific virus (21-25). These antigens therefore represent the viral proteins that are of major importance in regard to the vaccine problem. Encouraging results have been reported with the use of purified plasma membranes from virus-infected cells fot the prevention of Marek's disease (15). However, similar studies have not been reported for other herpesviruses. The reason for this is probably that many of the standard methods for isolating plasma membranes inactivate some herpesvirus-associated MA (26). It is therefore necessary to explore new approaches for the isolation of membrane preparations containing intact MA. A new method for isolating plasma membrane vesicles from cultured cells was recently reported by Scott (27). Exposure of cells in situ to various agents that share the common ability to bind to free sulfhydryl groups was shown to induce the formation of cell surface blebs and their release into the medium as free vesicles. Vesicles isolated from cultured fibroblasts were characterized ultrastructurally, biochemically, and functionally, and it was established that such vesicles were indeed derived from the plasma membrane. In this paper, we report results of preliminary studies that show that antigen-positive plasma membrane vesicles can be isolated from HVS-infected owl monkey kidney (OMK) cells with this technique, that the vesicles are free of detectable infectious virus and DNA, and that they can be used to induce neutralizing antibodies against HVS. MATERIALS AND METHODS Virus and Cell Culture. HVS passaged in OMK cells served as the virus inoculum for these experiments. The virus preparations had titers of approximately 105 plaque-forming units/ml as determined by the plaque assay for this virus described by Daniel et al. (28). The OMK cell line was cultured in the presence of RPMI 1640 medium containing 10% (vol/vol) heatinactivated fetal calf serum. Cell cultures were passaged twice weekly by trypsinization as previously described (20). Membrane Antigen Production. HVS was absorbed for 1 hr at 370 on confluent monolayers of OMK cells and then the cultures were incubated at 370 in an atmosphere of 5% C02/ 95% air, generally for 3 days. at this time, cultures usually showed extensive cytopathic effects characteristic of HVS. It was previously established that cells harvested when these changes were extensive contained a high percentage of MApositive cells as determined by membrane immunofluorescence

Abbreviations: HVS, Herpesvirus saimiri; MA, membrane antigens; OMK, owl monkey kidney; MF, membrane immunofluorescence; ADLC, antibody-dependent lymphocyte cytotoxicity; TNE buffer, 10 mM Tris/15 mM NaCI/1 mM EDTA.

(MF) (20).

Production of Plasma Membrane Vesicles. Monolayer cultures (75% confluent) of uninfected or HVS-infected OMK 2546

Immunology: Pearson and Scott cells showing virus-specific cytopathologic effects weredfid d to shed plasma membrane vesicles by the procedure recently described by Scott (27). Cultures were vigorously washed five times in RPMI 1640 to remove loosely adherent cells. Monolayers were then incubated for 2 hr at 370 in a vesiculant containing 25 mM formaldehyde/2 mM dithiothreitol prepared in RPMI 1640 medium. Whole cells that detached during this incubation period were removed by filtration through glass wool columns. Plasma membrane vesicles then were sedimented by centrifugation at 30,000 X g for 30 min at 4°. Only vesicle preparations found by phase microscopic examination to be free of contamination with cells and debris were processed further. Vesicle preparations, washed three times in 50 mM Tris-buffered saline, pH 7.4, were used in all the studies reported in this paper. Membrane Immunofluorescence Assay. The direct MF assay was performed on plasma membrane vesicles as previously described with whole cells, using a fluorescein-conjugated goat antiserum to human IgG (H- and L-chain specific; Hyland Laboratories, Los Angeles, CA) (20). The only modification was that vesicles were sedimented at 30,000 X g after treatment with serum and after the washes. The positive serum used for detecting MA-positive vesicles came from an owl monkey with HVS-induced lymphoma and had an MF titer of 320-640. The preparations were monitored for MA-positive vesicles with an American Optical model 2070 vertical fluorescence microscope. Inhibition of Antibody-Dependent Lymphocyte Cytotoxicity (ADLC). The ADLC assay was performed with 51Cr-labeled HVS-infected OMK cells as previously described in detail (29, 30). Serum from an owl monkey with HVS-induced lymphoma served as the source of antibody in this assay; this serum had an ADLC titer of 16,000-32,000. Baboon lymphocytes separated on Ficoll-Hypaque gradients (LSM Solution, Litton Bionetics, Inc., Kensington, MD) served as effector cells. [Cytotoxicity was calculated by subtracting the background (or spontaneous) release from the 51Cr release induced by the serum/lymphocyte mixtures divided by initial 51Cr incorporation minus spontaneous release times 100. 1 ADLC was calculated by subtracting the cytotoxicity value for lymphocytes incubated in the presence of normal serum from the cytotoxicity value in the presence of the immune serum. In the inhibition assays, two dilutions of the antibody-positive serum were absorbed twice with vesicles from uninfected OMK cells or from cells infected with HVS. The serum/vesicle mixtures were incubated at 370 for 1 hr and then overnight at 4°. The vesicles were removed by centrifugation at 30,000 X g and then the serum dilutions were tested for residual antibodies capable of mediating ADLC. The statistical significance of the decrease of ADLC activity after these absorptions in comparison to corresponding dilutions of unabsorbed serum was determined by Student's t test. Biochemical Determination. Plasma membrane vesicle protein concentration was determined routinely by the fluorescamine procedure (31) or by the Lowry assay (32); the results were comparable. Bovine serum albumin was used as the standard for these determinations. Electron Microscopy. Isolated plasma membrane pellets were fixed in 2.5% (vol/vol) glutaraldehyde/0.2 M phosphate buffer, pH 7.4, for 1 hr at room temperature and then overnight at 4°. Vesicles were postfixed in 2% (vol/vol) osmium tetroxide, dehydrated, and embedded in Epon. Specimens were stained en bloc in uranyl acetate. Thin sections were cut and stained in lead citrate. All specimens were examined in a Philips EM201 electron microscope.

Proc. Natl. Acad. Sci. -USA 74 (1977)

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l--itrunization of Nonhuman Primates. One baboon and three cottontop marmosets were immunized with three intramuscular inoculations of vesicles prepared from HVS-infected OMK cells; each animal received a total of approximately 1 mg of vesicle protein divided equally among the three injections. The first inoculum contained vesicles mixed with complete Freund's adjuvant. Three weeks later, the monkeys were injected with vesicles without adjuvant, and 2 weeks later they received a second booster injection. Blood was collected 7 days after the last immunization, and the serum was tested for antibodies to HVS-induced MA, late cytoplasmic antigens, and early antigens by immunofluorescence procedures (20, 33). The sera were also tested for neutralizing antibodies against 500 plaque-forming units of HVS. The last serum dilution to completely neutralize infectivity was taken to be the neutralization titer. All sera were exhaustively absorbed with uninfected OMK cells before the serological studies were performed. Test for Infectious HVS. Experiments to determine whether infectious HVS was present in the vesicle preparations included cocultivation of vesicles with OMK cells grown in Costar 24-well tissue culture cluster dishes. The cultures were followed for signs of HVS-induced cytopathic effects for a minimum of 2 weeks. The cultures were also stained for the presence of viral antigens by fixed cell immunofluorescence (33) to determine whether viral antigen expression could be detected in the cocultures. For bioassay, cottontop marmosets were injected with approximately 1.0 mg of HVS plasma membrane vesicle protein and were observed for signs of malignant disease. DNA Determination. OMK cell monolayers were infected with HVS, and 24 hr thereafter [3H]thymidine (25 jACi/ml; 23 Ci/mmol; New England Nuclear, Boston, MA) was added to each of 15 175-cm2 flasks. Cultures were labeled for 48 hr. The specific activity of cellular DNA was determined after the preparation of vesicles from these cells. Intact cells from individual flasks were removed by trypsinization. Cells lysed with 1.0% (vol/vol) sodium dodecyl sulfate in 10 mM Tris/15 mM NaCI/1 mM EDTA (TNE buffer) were extracted twice with phenol/cresol and 4% (vol/vol) isoamyl alcohol (34). An aliquot was precipitated with trichloroacetic acid, filtered, and assayed for radioactivity. The remainder was precipitated with ethanol and the cellular DNA was sedimented by centrifugation at 296,000 X g in a SW-41 rotor.. This material was resuspended in 0.5 M NaOH and incubated at 370 overnight. It was then neutralized, extracted with phenol/cresol, and again ethanolprecipitated. The precipitate was collected by centrifugation as above and resuspended in 300 jul of sterile water. The absorbance at 280 and 260 nm was recorded and the amount of DNA present was determined. This sample was then precipitated with trichloroacetic acid, filtered, and assayed for ra-

dioactivity. -Plasma membrane vesicles were lysed with 0.5% sodium dodecyl sulfate in TNE buffer. An aliquot was precipitated with trichloroacetic acid, filtered, and assayed for radioactivity. The remainder was extracted as above and ethanol-precipitated. The precipitate, collected by centrifugation, was resuspended in 20 jul of sterile water and divided into four equal portions that were treated as follows: (i) diluted to 1 ml with TNE buffer and left untreated; (ii) diluted in 1 ml of 0.3 M NaCI/0.03 M sodium citrate, pH 8.6, to which 50,ug of RNase A (50 units/mg; P-L Biochem, Milwaukee, WI) was added; (iii) diluted with 1 ml of 0.01 M Tris-HCI, pH 7.0/0.15 M NaCI/3 mM MgCl2 to which 50 ,ug of DNase I (2800 units/mg; Schwartz/Mann, Orangeburg, NY) was added. Each of these three portions was incubated at 370 for 45 min and then precipitated with trichloroacetic acid, filtered, and assayed for radioactivity. The

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Isolation of virus-free Herpesvirus saimiri antigen-positive plasma membrane vesicles.

Proc. Natl. Acad. Sci. USA Vol. 74, No. 6, pp. 2546-2550, June 1977 Immunology Isolation of virus-free Herpesvirus saimiri antigen-positive plasma m...
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