VIROLOGY
189, 266-273
(1992)
Flow Cytometric Analysis of African Swine Fever Virus-Induced Plasma Membrane Proteins and Their Humoral Immune Response in Infected Pigs CARLOS ALCARAZ,* ALBERT0 ALVAREZ,t
AND
JOSi M. ESCRIBANO*”
*Departamento de Sanidad Animal, lnstituto National de Investigacih y Tecnologla Agraria y Alimentaria (INIA), Embajadores 68, 280 12 Madrid, Spain; tCentro de Citometria de flujo, Universidad Complutense de Madrid, facultad de Farmacia, Madrid, Spain Received April 18, 199 1; accepted April 6, 1992 African swine fever (ASF) virus-induced plasma membrane proteins may contribute to the protective immune response against the disease since they can be involved in the antibody-mediated lysis of infected cells. In this study we describe the regulation of ASF virus-induced plasma membrane protein expression and its antibody induction in pigs after viral infection by flow cytometric analysis. More than 80% of infected cells contained viral antigens on the surface membranes at 6 hr postinfection (hpi), and the relative amount of viral antigen expression was increased at 12 and 20 hpi. The kinetics of individual viral protein expression on cell surfaces was studied by a collection of monospecific antibodies directed against the six viral plasma membrane proteins ~12, ~15, ~16, ~23.5, ~30, and ~35. Most of these proteins were expressed at 6 hpi, with tlie exception of ~35, which was first detected at 12 hpi. The percentage of cells expressing each antigen at different hpi was also determined. The immune response against virus-induced plasma membrane proteins in pigs infected with an attenuated ASF virus strain was studied. Antibodies against viral epitopes exposed on plasma membranes reached a plateau at 20 days postinfection (dpi). The relative amount of antibodies induced during infection with these specificities was not directly related to the antibody titer of the sera. Sera obtained at 20 and 40 dpi contained antibodies against most of the viral plasma membrane proteins and were most efficient in o 1992 Academic PESS, inc. recognition of viral antigens exposed on the surface of infected cells at early times.
INTRODUCTION
resistant to challenge with homologous and, occasionally, heterologous isolates (Malmquist, 1963; Manso Ribeiro eta/., 1963; Ruiz-Gonzalvo eta/., 1986). At present, the immunological mechanisms involved in protection have not been demonstrated, although humoral and cellular immune responses have been described after ASFV infection. These include infectioninhibition antibodies (Ruiz-Gonzalvo et al., 1986a,b), antibody-dependent cell-mediated cytotoxicity (ADCC) (Norley and Wardley, 1982; Wardley eta/., 1985), complement-dependent antibody-mediated cytotoxicity (CDAC) (Wardley et al., 1985) and ASFV-specific cytotoxic T cells (Norley and Wardley, 1984; Martins et al,, 1988). Some of them are mediated by virus-induced proteins on the surface of infected cells. In addition, experiments in which antibodies from pigs that survive ASFV infection are transferred to susceptible pigs have demonstrated a partial resistance to virus inoculation of the recipient pigs with a decrease in the virus titer in blood (Schlafer et a/., 1984a,b; Wardley et a/., 1985). Previous studies have shown differences in antibody induction between pigs immunized with ASFV-soluble antigens, after which about 10% are clinically resistant to challenge with homologous virulent virus, and pigs infected with live attenuated virus, after which 100% are protected to the virulent virus (Alcaraz et a/., 1989; Alcaraz and Escribano, unpublished data). The differences in antibody response represent qualitative and
African swine fever virus (ASFV) is an icosahedral cytoplasmic deoxyvirus that infects porcine species (for review see Viiluela, 1985). ASFV DNA is a linear double-stranded (Enjuanes et a/., 1976) molecule with covalently closed ends (Or-tin eta/., 1979) and a molecular weight (MW) of about 10’ (Enjuanes et al., 1976). It codes for the synthesis of about 100 polypeptides in cell lines (Esteves et a/., 1986; Santa&n and ViAuela, 1986; Urzainqui et a/., 1987) and 86 in pig macrophages (Alcaraz et a/., 1992) ranging in MW from 9.5K to 243K. Ten of these virus-induced proteins are expressed on the surface of infected pig macrophages (Alcaraz et a/., 1989). The same proteins were detected on the surface of a monkey stable (MS) cell line after infection with the virus (Alcaraz and Escribano, unpublished data). Attempts to use inactivated viruses or antigens to induce protection against ASFV have largely failed (DeTray, 1963; Stone and Hess, 1967; Tabares et al., 1981). The most efficient methods of inducing protection against the infection use live attenuated viruses (Malmquist, 1963; Thomson et a/., 1979; Ruiz-Gonzalvo et al., 1986). Most of the pigs recovered from infection with an attenuated ASFV isolate are clinically ’ To whom reprint requests should be addressed. 0042-6822/92
$5.00
Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
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quantitative differences in protein recognition. Some of these are virus-induced plasma membrane proteins, which have been shown to induce protective antibodies in vivo in other viral diseases (Leconte el al., 1987; Falgout et al., 1990; Putnak and Schlessinger, 1990). The purpose of the present study was to characterize cell surface-expressed viral proteins and the humoral immune response elicited against these in pigs immunized with an attenuated ASFV isolate.
MATERIALS Cells, viruses,
AND METHODS
and sera
For in vitro assays, the Spain-70 (E70)strain of ASFV was used after 46 passages in a monkey stable (MS) cell line (Alcaraz et a/., 1989) at a multiplicity of infection of 5. For pig inoculation the Spain-75 (E75) strain was used after four passages in CVl cells (ATC CCL70) (ET5 CVl-4). This viral strain, when inoculated orally or intramuscularly, confers protection against subsequent challenge with homologous virulent virus (Ruiz-Gonzalvo et al., 1986; Alcaraz and Escribano, unpublished data). Three NIH minipigs (Sachs et al., 1976) of SLA”” weighing about 20 kg and numbered as 1, 2, and 3 were infected orally with 2 X 1O3 50% haemadsorbing dose (HAD,,) of E,, CVl-4 virus. Two minipigs numbered as 4 and 5, and having the same characteristics, were inoculated intramuscularly with 1O3 HAD,, of the same virus to collet sera. Pig 5 died at 6 days postinfection (dpi) of ASF. The surviving pigs (1, 2, 3, and 4) were bled before inoculation and at Postinoculation Days 5, 1 1, 20, and 40. Serum obtained from pig 4 on Postinoculation Day 20 was used as hyperimmune ASFV serum. Serum obtained from the same pig on Postinoculation Day 40 was used for affinity purification of antibodies from Western blots.
Polyacrylamide gel electrophoresis Western blotting
and
All procedures for electrophoresis were as previously described in acrylamide-l\l, N’diallyltartardiamide (DATD) gels (Escribano and Tabares, 1987). Proteins used for Western blotting analysis were those contained in the cytoplasmic soluble antigen (Escribano et al., 1989) obtained by treatment of infected cells with the detergent nonidet P40 in presence of high ionic strength. Proteins were resolved on 17% acrylamide gels and transferred to nitrocellulose filters. Transfer conditions and subsequent analysis were the same as previously described (Alcaraz et a/., 1990).
Affinity
purification
of antibodies
267
from Western blots
Antibodies that bind to a given polypeptide band were purified from polyclonal sera by a simple modification of the Western blotting technique (Olmsted, 1981; Smith and Fisher, 1984). Cytoplasmic ASFV soluble antigen obtained as described (Escribano et a/., 1989) from 20-30 X lo6 infected cells, and 10” cpm of the same antigen labeled with [35S]methionine (Alcarazetal., 1990) were loaded into a large preparative well of a 17% acrylamide gel, electrophoretically separated, and then transferred to a nitrocellulose filter. The filter was dried and exposed to X-ray film to localize the ASFV-induced proteins. After protein localization, areas of the blot that contained the antigen were excised using a sharp scalpel. For affinity purification of antibodies, strips containing the different antigens were incubated with a hyperimmune serum after blocking with 2% nonfat dry milk. The antiserum was used diluted at 1: 10 in washing buffer (2% nonfat dry milk, 0.05% Tween in PBS, pH 7.2). Strips were washed six times with washing buffer and monospecific antibodies were eluted with 0.1 M glycine, pH 2.8, for 15 min with agitation. The pH of the eluted antibodies was neutralized with 1:5 volume 0.1 M NaOH in 50 mM Tris-HCI, pH 7. The strips were extensively washed and used more than five times. Antibodies were dialyzed against distilled water, lyophiIized, and resuspended in about 1 :lO of the original volume.
lmmunofluorescence
and flow cytometric
analysis
Separate ASFV-infected MS cells at different hpi obtained after incubation with 10 mM EDTA in PBS, pH 7.2, at 4” were washed with staining buffer. Cells were then incubated with either hyperimmune serum at 1:40 dilution or nondiluted monospecific antiserum (previously determined as saturating antibody concentrations) for 30 min at 4”. Then, cells were washed twice with staining buffer and incubated with a second FITC rabbit anti-swine IgG at 1:30 dilution for 30 min at 4”. After two more washes with staining buffer, cells were fixed with 1% paraformaldehyde in staining buffer. Stained cells were run in a FACStar PLUS (Becton Dickinson, San Jose, CA) flow cytometer equipped with a 2-watt argon laser tuned to 488 nm. Data were analyzed with FACStar PLUS Research Software (BectonDickinson) in a C30 computer. LYSYS II C32 Software (Becton-Dickinson) was used to obtain Fig. 1 B. Fluorescence data were collected by using logarithmic amplification on 5000 cells as determined by forward light scatter intensity. One-color fluorescence data were displayed as immunofluorescence profiles in which log
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FSCFIG. 1. Expression of ASFV-specified antigens on the plasma membranes of infected cells. (A) Flow cytometric analysis of ASFV antigen expression. x-axis represents the size of sample (FSC), whereas the y-axis represents the relative fluorescence intensity (log-scale) of the cell population at 0 hpi (Oh) and 6 hpi (6h). (B) Flow cytometric analysis of the relative cell number that presents different fluorescence intensity at 0, 6, 12, and 20 hpi.
fluorescence intensity was plotted on the x axis and cell number on they axis.
RESULTS Cell surface expression of ASFV-induced plasma membrane proteins By flow cytometric analysis of unfixed cells incubated with hyperimmune serum at 0, 6, 12, and 20 hpi over 80% of infected cells showed specific fluorescence at 6 hpi (Fig. 1A). This indicated that one or more ASFV antigens were expressed on the cell surface at this time point. Infected cells increased their relative fluorescence at 12 and 20 hpi with respect to that obtained at 6 hpi (Fig. 1 B). No significant differences in the percentage of cells that expressed viral antigens on their surfaces-over 90%-or their relative fluorescence intensities were found between 12 and 20 hpi. This result suggested maximum viral antigen expression at about 12 hpi.
Kinetics of individual protein expression on the cell surface Affinity-purified monospecific antisera against six virus-induced plasma membrane proteins with relative molecular weights of 12K, 15K, 16K, 23.5K, 30K, and 35K, and another against the major viral structural protein of 73K, were obtained from a recovered pig serum after ASFV infection as described in materials and methods. All antisera reacted with only one protein by Western blotting, with the exception of the antiserum raised against the 35K polypeptide, which also reacted
AND ESCRIBANO
with a higher molecular weight polypeptide of about 60K, probably a precursor protein (Fig. 2). Seven antisera were used in a flow cytometric analysis of infected cells at 0, 6, 12, and 20 hpi. Each antiserum reacted with infected unfixed cells as early as 6 hpi with the exception of antiserum against ~35, which first gave significant fluorescence at 12 hpi, and antiserum against ~73, which did not react at any time after infection (Fig. 3). The percentage of infected cells that expressed each protein at different hpi is shown in Fig. 4. Protein pl2 was detected on the cell surface in at least 91% of infected cells. Polypeptides ~15, ~16, ~23.5, ~30, and p35 were expressed in 78, 85, 83, 82, and 78% of infected cells, respectively. The maximum percentage of infected cells that expressed proteins ~12, ~15, ~16, ~23.5, and p30 was obtained at 6 hpi and did not vary significantly at 12 and 20 hpi. Antiserum against p35 reacted with the maximum percentage of unfixed infected cells from 12 hpi (Fig. 4).
Immune response against virus-induced membrane proteins
plasma
Pigs 1, 2, and 3, orally inoculated with attenuated ASFV E,, CVl-4 strain, and pig 4 which survived intra-
I2345678
-73K
-35 K -3OK -23.5
K
-16K -15K -12K FIG. 2. Affinity purification of antibodies against seven virus-induced proteins. Antibodies against the virus-specified proteins p73 (lane l), p35 (lane 2) p30 (lane 3) ~23.5 (lane 4) pl6 (lane 5) pl5 (lane 6) and pl2 (lane 7) were affinity purified from an hyperimmune serum. Their specificity was characterized by Western blotting on nitrocellulose strips containing a soluble antigen obtained from infected cells. The hyperimmune serum used in the affinity purification of monospecific antibodies was used as control in Western blotting (lane 8).
AFRICAN
SWINE FEVER-INDUCED
PI2
P23.5
1
P30
P73
I
I
-
Rd0tive
fluorescence
intensity -
FIG. 3. Krnetrc expression of rndrvrdual ASFV-specified plasma membrane proterns. These are fluorescence histograms that result from flow cytometric analyses of unfixed ASFV-infected cells at 0 hpr (l), 6 hpi (2) 12 hpr (3) and 20 hpr (4) incubated with six monospecific antisera against viral plasma membrane proterns (~12, ~15, ~16, ~23.5, ~30, and ~35) plus a FITC rabbit anti-swine serum. The serum against viral protein p73 was used as a representatrve control of internal nonexpressed protein on the surface of Infected cells, Fluorescence Intensity is presented (log-scale) on the x-axis, whereas the y-axis represents the relative number of cells.
muscular inoculation of the same virus; were used to study antibody induction against viral plasma membrane proteins. Pigs 1, 2, and 3 had an inapparent disease and shorter viraemias, 15-20 days, than pig 4, which showed appreciable clinical signs and 30 days of viraemia (data not shown). Moreover, the virus titers in peripheral blood were at least four logarithms higher in pig 4 than in pigs 1, 2, and 3 (data not shown). These differences in viraemias and clinical signs, depending on the inoculation method used, probably resulted in pig 4 presenting higher specific antibody titer at 40 dpi than pigs 1, 2, and 3 (Figs. 5A and 7A). All pigs were bled before inoculation and on Postinoculation Days 5, 11, 20, and 40. Antibodies against virus-induced plasma membrane proteins were detected from Postinoculation Day 1 1 by Western blotting in all pigs. Antibodies against ~12, ~23.5, and p30 were detected on Day 11 postinfection. At 20 dpi, antibodies against all
PLASMA
MEMBRANE
269
PROTEINS
the previously characterized plasma membrane proteins were detected in pigs 1, 2, and 3, but pig 4 needed 40 days to induce antibodies against protein p14 (Fig. 5A; Fig. 7B). Interestingly, protein pl2 was less reactive with antibodies obtained at Days 20 and 40 after infection than those obtained at Day 1 1 in pig 3 (data not shown) and in Pig 4 (Fig. 7B), but not in pig 1 (Fig. 5A). These sera were also used in flow cytometric analysis of cells at 0, 6, 12, and 20 hpi. We found specific fluorescence in unfixed infected cells using sera from Days 1 1 to 40 after infection in all inoculated pigs (Figs. 5B, 6, 7A). However, early viral proteins, expressed on the cell surfaces before newly formed infective virus particles could be released from infected cells after potential antibody-mediated ceil lysis, reacted significantly only with sera obtained after 20 dpi. Sera obtained from both orally and intramuscularly inoculated pigs at 20 and 40 dpi recognized similar percentages of infected cells and also showed similar behavior relative to the time after infection when antibodies recognized infected cells (Figs. 6, 7A). Apparently, antibodies to viral epitopes exposed on the surface of infected cells reached a plateau at about 20 dpi In all inoculated pigs, even if the specific antibody titer against the virus was increased at later dpi, as shown in pig 4 (Fig. 7A).
DISCUSSION Virus-induced plasma membrane proteins can mediate in vitro the interaction of infected cells with cell-mediated and humoral components of the immune re1001
0 Hours
6
I2 after
20 infection
FIG. 4. Percentage of ASFV-Infected cells expressing different viral anttgens on the cell surface. This figure, based on the flow cytometry results shown in Fig. 3, represents the percentage of fluorescent cells obtained at 0, 6, 12, and 20 hpr after incubation with the monospecific antisera against vrral specrfied proterns pl2 (A), pl5 (+), pl6 (m), ~23.5 (A), p30 (II), p35 (0) and p73 (0) plus FITC rabbrt antrswine serum. Data are expressed as the arithmetic mean of three fluorescence assays carried out with each monospecifrc serum; the standard deviatron was lower than 4% in every case.
270
ALCARAZ,
ALVAREZ,
AND ESCRIBANO
B dpi ----P 0
9.6.T--
5
11
20
40
.zii 6.. 8m 6.- 4-3-2 -I --
Days after infection
Raktive
cell number
FIG. 5. Antibody response to ASFV-specified and plasma membrane proteins in orally inoculated prgs. (A) (I) Analysis of the specific antibody ELISA titer in three ASFV orally inoculated pigs numbered as 1 (0) 2 (m), and 3 (A) measured by ELISA titer obtained at different dpi. (II) Western blotting reactions on nitrocellulose strips containing soluble cytoplasmic antigen from infected cells with sera obtained from pig 1 before inoculation and at Postinoculation Days 5, 11, 20, and 40. (B) Antibody response to virus-specified plasma membrane proteins in pig 1, representative of the orally inoculated pigs. The relative number of fluorescent cells was analyzed by flow cytometry at 0, 6, 12, and 20 hpi when incubated with pig sera obtained before inoculation (0) and at 5, 11, 20, and 40 dpi plus FITC rabbit anti-swine serum. In fluorescence histograms, fluorescence intensity is represented (log-scale) on the x-axis, whereas the y-axis represents the relative number of cells.
sponse elicited during viral infections (Mallon et al., 1985; Goudsmit et al., 1988; Falgout et al., 1990; Laviada eta/., 1990; Schlesinger et al., 1990). If an in viva correlate of this phenomenon can be established, the virus specified cell-surface antigens could play an important role during the immune response in ASFV-infected pigs.
I
I
0
6 I2 Hours after infection
20
FIG.6. Average percentage of ASFV-infected cells expressing viral antigens on the cell surface which react with sera obtained from the three orally infected pigs at different days after infection. This figure shows the percentage of fluorescent ASFV-infected cells at 0, 6, 12, and 20 hpi after reaction with sera from the infected pigs obtained at 0 (0) 5 (m), 11 (A), 20 (Cl), and 40 dpi (A) plus FITC rabbit anti-swine serum.
The results of our experiments confirm and extend the observation that ASFV antigens are expressed on the plasma membrane of infected cells. The data provide evidence that the majority of viral antigens on infected cell surfaces are expressed at early times after infection, before newly infective virus is produced. Previous studies have indicated that all proteins detected on the cell surface are synthesized in the presence of phosphonoacetic acid, which only permits synthesis of proteins before viral DNA replication (Alcaraz et al., 1989). However, in this study, p35 was expressed in large quantities later than the other antigens. Evidence for a 32K polypeptide that appears later in virus-infected Vero cell membranes than other viral antigens has been previously described (Santa&n and Vifiuela, 1986). In viruses with similar genomic organization to ASFV, such as vaccinia virus, about nine virus-induced proteins are expressed on the surface of infected cells (Mallon and Holowczak, 1986). The orthopoxvirusspecified antigens that become associated with the plasma membrane of infected cells can be divided into three categories (Oie and Ichihashi, 1981). Two of these are the “early” viral antigens expressed prior to the onset of viral DNA synthesis (Ueda et al., 1969, 1972; Koszinowski et al., 1976) and the “late” viral antigens which are expressed during or after viral DNA replication is completed (Weintraub and Dales, 1974; Dales and Oldstone, 1982). The third group consists of
AFRICAN
SWINE FEVER-INDUCED
PLASMA
MEMBRANE
PROTEINS
0 5 II
271
2040
= I-73,
Days after infection
w
. ,*
--l6K B-l5K
_
=i-l2K
Hours after infection FIG. 7. Antibody response to ASFV-specified and plasma membrane proterns in a rntramuscularly Inoculated prg. (A) Comparison of the antibody titer in ELISA (top) and vrral plasma membrane recognition at 0, 6, 12, and 20 hpi by sera obtained at 5 (0) 1 1 (m), 20 (e), and 40 (A) dpt by flow cytometry (bottom). (B) Western blotting reactions on nitrocellulose strips contarnrng soluble cytoplasmic ASFV antigens from infected cells with sera obtained from pig 4 intramuscularly inoculated at time before inoculation (lane 0) and at 5 (lane 5), 1 1 (lane 1 l), 20 (lane 20), and 40 dpr (lane 40).
virion antigens transferred to the plasma membrane as a result of fusion of the viral envelope with the plasma membrane during adsorption and penetration of virus particles into cells (Oie and Ichihashi, 1981). This last category of ASFV proteins was not studied in the present work. Proteins ~12, ~15, ~16, ~23.5, and p30 can be classified as “early” expressed proteins and protein p35 as a “late” viral plasma membrane antigen. Complement-dependent antibody-mediated cytotoxicity (CDAC) (Norley and Wardley, 1982) and antibodydependent cell-mediated cytotoxicity (ADCC) (Wardley et a/., 1985) have been described in African swine fever. These immune mechanisms can be elicited by one or more viral plasma membrane proteins (Alcaraz et al., 1989). Antibodies produced during ASFV infection partially protected pigs that were intraperitoneally passively immunized (Schlaferetal., 1984b; Wardleyet al., 1985) or suckling piglets (Schlafer et al., 1984a,b) against the effects of virulent ASFV challenge. Protection, which was exemplified by a reduction in pyrexia and viraemia plus an increased survival time, appeared to be mediated through the effects of CDAC and ADCC immune mechanisms (Wardley et al., 1985). These authors suggested that reduction in viraemia was associated with complement lysis, whereas protection was conferred by ADCC. Effective lysis of infected cells mediated by antibodies reacting with viral plasma membrane proteins is determined by two factors: (i) viral antigen concentration on the cell surface and (ii) titer and isotype of the
antibodies reacting with the viral antigens. For most virus-induced plasma membrane proteins, the quantity of antigen expressed on the surface of infected cells reached a plateau between 6 and 12 hpi. By using sera from infected pigs obtained at 20 and 40 dpi, the fluorescence intensities and percentages of positive cells at 6, 12, and 20 hpi were very similar. This indicates that viral antigen concentration sufficient to elicit lysis of infected cells mediated by antibodies could be reached very soon after infection, prior to formation of new infective viral particles. Development of antibody capable of initiating CDAC and ADCC has been described as a relatively late event in infected pigs (Norley and Wardley, 1982). This supports our results obtained after analysis of the antibody response against virus-induced plasma membrane proteins. We have shown that infected pigs need about 20 days after infection to develop optimal recognition of viral antigens expressed on the cell surfaces at early times after infection. However, viral plasma membrane proteins such as pl2 and p30 induced a considerable amount of antibody, detected by Western blotting, as early as 11 dpi and could play an important role in the lysis of infected cells in the first stages of the disease. No experiments have been carried out in naive pigs to demonstrate the protective efficiency in viva after transfer of antibodies obtained at different times after infection. However, in vitro, antibodies obtained at 1415 dpi but not at 5-7 dpi were effective in fixing complement to a degree sufficient for cell lysis (Norley and
272
ALCARAZ,
ALVAREZ,
Wardley, 1982). At 14-l 5 dpi the same authors obtained the maximum percentage of complement-mediated lysis, suggesting that the onset of this mechanism is not simply the result of increasing levels of specific antibody in the serum. We found that antibodies in sera obtained at 20 and 40 dpi are the most efficient with respect to the relative number of infected cells they recognize and the fluorescence intensity they provide to the cells at 6 hpi. African swine fever virus does not produce easily detectable neutralizing antibodies. However, since there is evidence of ASFV challenge protection by antibodies, we have focused attention on structural and nonstructural ASFV-induced proteins which are expressed on the surface of infected cells. Recent studies with flaviviruses have demonstrated that protective immunity can be elicited by a nonstructural protein, NSl, which is expressed on the cell surface (Falgout et a/., 1990; Putnak and Schlessinger, 1990). Similar results were obtained with plasma membrane proteins in other viral diseases, such as mouse hepatitis virus type 3 (Leconte et a/., 1987). Experiments to elucidate the relevance of the characterized ASFV plasma membrane proteins in the immunological mechanisms involved in protection against this disease are underway. ACKNOWLEDGMENTS This work was supported by a grant from the lnstituto National de Investigaci6n y Tecnologfa Agraria y Alimentaria (INIA). We thank Doctors D.L. Rock, D.P. Schenkein, F. Ruiz-Gonzalvo, and M. De Diego for useful discussions.
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RUIZ-GONZALVO,F., CABALLERO,C., MARTINEZ, J., and CARNERO, M. E. (198613). Neutralization of African swine fever virus by sera from African swine fever-resistant pigs. Am. 1. Vet. Res. 47, 18581962. SACHS, D. H., LEIGHT, G., CONE, J.. SCHWARZ, S., STUART, L.. and ROSENBERG,S. (1976). Transplantation in miniature swine. I. Fixation of the major histocompatlbillty complex. Transplantation 22, 559-567. SANTAR~N, J. F., and VIKJUELA,E. (1986). African swine fever virus-induced polypeptides in vero cells. Vks Res. 5, 391-405. SCHLAFER, D. H., MCVICAR, 1. W., and MEBUS, C. A. (1984a). African swine fever convalescent sows: Subsequent pregnancy and the effect of colostral antibody on challenge inoculation of their pigs. Am. 1. ve/et.Res. 45, 1361-1366. SCHLAFER, D. H., MEBUS, C. A., and MCVICAR, J. W. (1984b). African swine fever in neonatal pigs: Passively acquired protection from colostrum or serum of recovered pigs. Am. J. Vet. Res. 45, 13671372. SCHLESINGER,J. J., BRANDRISS,M. W., PUTNAK,J. R., and WALSH, E. E. (1990). Cell surface expression of yellow fever virus non-structural glycoprotein NSl : Consequences of interaction with antibody. /. Gem Viral. 71, 593-599. SMITH, D. E., and FISHER, P. A. (1984). Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: Application of an Improved method for affinity purification of anti-
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bodies using polypeptldes immobilized on nitrocellulose blots. J. Cell Biol. 99, 20-28. STONE, S. S., and HESS, W. R. (1967). Antibody response to inactivated preparattons of ASFV. Am. 1. Vet. Res. 28, 475-481. TABAR~S, E., FERNANDEZ, M., SALVADOR-TEMPRANO, E., CARNERO, M. E., and SANCHEZ-B• TIJA, C. (1981). A reliable enzyme kinked immunosorbent assay for African swine fever using the major structural protein as antigenic reagent. Arch. Viral. 70, 297-300. THOMSON, G. R., GAINARU, M. D., and VAN DELLEN, A. F. (1979). ASF: Pathogenicity and lmmunogenlcity of 2 non-haemadsorbtng VIruses. Onderstepoort J. Vet. Res. 47, 149-l 54. &DA, Y., ITO, M., and TAGAYA, J. (1969). A specific surface antigen induced by poxvirus. Virology 38, 180- 182. UEDA, Y., TAGAYA, J., AMANO, H., and ITO, M. (1972). Studies on the early antigens Induced by vacclnla virus. Virology 49, 794-800. URZAINQUI,A., TABAR~S, E., and CARRASCO,L. (1987). Proteins synthesized in African swine fever virus-infected cells analyzed by two-dim mensional gel electrophoresis. Virology 160, 286.-291. VKJUELA, E. (1985). African swine fever virus. Curr. Top. Microbial. Immunoi. 116, 151-170. WARDLEY, R. C., NORLEY, S. G., WILKINSON, P. J., and WILLIAMS, S. (1985). The role of antibody in protection against African swine fever virus. Vet. lmmunol. Immunopathol. 9, 201-212. WEINTRAUB.S., and DALES, S. (1974). Biogenests of poxvirus: Genetically controlled modifications of structural and functional components of the plasma membrane. Virology 60, 96-l 27.
189, 266-273
(1992)
Flow Cytometric Analysis of African Swine Fever Virus-Induced Plasma Membrane Proteins and Their Humoral Immune Response in Infected Pigs CARLOS ALCARAZ,* ALBERT0 ALVAREZ,t
AND
JOSi M. ESCRIBANO*”
*Departamento de Sanidad Animal, lnstituto National de Investigacih y Tecnologla Agraria y Alimentaria (INIA), Embajadores 68, 280 12 Madrid, Spain; tCentro de Citometria de flujo, Universidad Complutense de Madrid, facultad de Farmacia, Madrid, Spain Received April 18, 199 1; accepted April 6, 1992 African swine fever (ASF) virus-induced plasma membrane proteins may contribute to the protective immune response against the disease since they can be involved in the antibody-mediated lysis of infected cells. In this study we describe the regulation of ASF virus-induced plasma membrane protein expression and its antibody induction in pigs after viral infection by flow cytometric analysis. More than 80% of infected cells contained viral antigens on the surface membranes at 6 hr postinfection (hpi), and the relative amount of viral antigen expression was increased at 12 and 20 hpi. The kinetics of individual viral protein expression on cell surfaces was studied by a collection of monospecific antibodies directed against the six viral plasma membrane proteins ~12, ~15, ~16, ~23.5, ~30, and ~35. Most of these proteins were expressed at 6 hpi, with tlie exception of ~35, which was first detected at 12 hpi. The percentage of cells expressing each antigen at different hpi was also determined. The immune response against virus-induced plasma membrane proteins in pigs infected with an attenuated ASF virus strain was studied. Antibodies against viral epitopes exposed on plasma membranes reached a plateau at 20 days postinfection (dpi). The relative amount of antibodies induced during infection with these specificities was not directly related to the antibody titer of the sera. Sera obtained at 20 and 40 dpi contained antibodies against most of the viral plasma membrane proteins and were most efficient in o 1992 Academic PESS, inc. recognition of viral antigens exposed on the surface of infected cells at early times.
INTRODUCTION
resistant to challenge with homologous and, occasionally, heterologous isolates (Malmquist, 1963; Manso Ribeiro eta/., 1963; Ruiz-Gonzalvo eta/., 1986). At present, the immunological mechanisms involved in protection have not been demonstrated, although humoral and cellular immune responses have been described after ASFV infection. These include infectioninhibition antibodies (Ruiz-Gonzalvo et al., 1986a,b), antibody-dependent cell-mediated cytotoxicity (ADCC) (Norley and Wardley, 1982; Wardley eta/., 1985), complement-dependent antibody-mediated cytotoxicity (CDAC) (Wardley et al., 1985) and ASFV-specific cytotoxic T cells (Norley and Wardley, 1984; Martins et al,, 1988). Some of them are mediated by virus-induced proteins on the surface of infected cells. In addition, experiments in which antibodies from pigs that survive ASFV infection are transferred to susceptible pigs have demonstrated a partial resistance to virus inoculation of the recipient pigs with a decrease in the virus titer in blood (Schlafer et a/., 1984a,b; Wardley et a/., 1985). Previous studies have shown differences in antibody induction between pigs immunized with ASFV-soluble antigens, after which about 10% are clinically resistant to challenge with homologous virulent virus, and pigs infected with live attenuated virus, after which 100% are protected to the virulent virus (Alcaraz et a/., 1989; Alcaraz and Escribano, unpublished data). The differences in antibody response represent qualitative and
African swine fever virus (ASFV) is an icosahedral cytoplasmic deoxyvirus that infects porcine species (for review see Viiluela, 1985). ASFV DNA is a linear double-stranded (Enjuanes et a/., 1976) molecule with covalently closed ends (Or-tin eta/., 1979) and a molecular weight (MW) of about 10’ (Enjuanes et al., 1976). It codes for the synthesis of about 100 polypeptides in cell lines (Esteves et a/., 1986; Santa&n and ViAuela, 1986; Urzainqui et a/., 1987) and 86 in pig macrophages (Alcaraz et a/., 1992) ranging in MW from 9.5K to 243K. Ten of these virus-induced proteins are expressed on the surface of infected pig macrophages (Alcaraz et a/., 1989). The same proteins were detected on the surface of a monkey stable (MS) cell line after infection with the virus (Alcaraz and Escribano, unpublished data). Attempts to use inactivated viruses or antigens to induce protection against ASFV have largely failed (DeTray, 1963; Stone and Hess, 1967; Tabares et al., 1981). The most efficient methods of inducing protection against the infection use live attenuated viruses (Malmquist, 1963; Thomson et a/., 1979; Ruiz-Gonzalvo et al., 1986). Most of the pigs recovered from infection with an attenuated ASFV isolate are clinically ’ To whom reprint requests should be addressed. 0042-6822/92
$5.00
Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
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quantitative differences in protein recognition. Some of these are virus-induced plasma membrane proteins, which have been shown to induce protective antibodies in vivo in other viral diseases (Leconte el al., 1987; Falgout et al., 1990; Putnak and Schlessinger, 1990). The purpose of the present study was to characterize cell surface-expressed viral proteins and the humoral immune response elicited against these in pigs immunized with an attenuated ASFV isolate.
MATERIALS Cells, viruses,
AND METHODS
and sera
For in vitro assays, the Spain-70 (E70)strain of ASFV was used after 46 passages in a monkey stable (MS) cell line (Alcaraz et a/., 1989) at a multiplicity of infection of 5. For pig inoculation the Spain-75 (E75) strain was used after four passages in CVl cells (ATC CCL70) (ET5 CVl-4). This viral strain, when inoculated orally or intramuscularly, confers protection against subsequent challenge with homologous virulent virus (Ruiz-Gonzalvo et al., 1986; Alcaraz and Escribano, unpublished data). Three NIH minipigs (Sachs et al., 1976) of SLA”” weighing about 20 kg and numbered as 1, 2, and 3 were infected orally with 2 X 1O3 50% haemadsorbing dose (HAD,,) of E,, CVl-4 virus. Two minipigs numbered as 4 and 5, and having the same characteristics, were inoculated intramuscularly with 1O3 HAD,, of the same virus to collet sera. Pig 5 died at 6 days postinfection (dpi) of ASF. The surviving pigs (1, 2, 3, and 4) were bled before inoculation and at Postinoculation Days 5, 1 1, 20, and 40. Serum obtained from pig 4 on Postinoculation Day 20 was used as hyperimmune ASFV serum. Serum obtained from the same pig on Postinoculation Day 40 was used for affinity purification of antibodies from Western blots.
Polyacrylamide gel electrophoresis Western blotting
and
All procedures for electrophoresis were as previously described in acrylamide-l\l, N’diallyltartardiamide (DATD) gels (Escribano and Tabares, 1987). Proteins used for Western blotting analysis were those contained in the cytoplasmic soluble antigen (Escribano et al., 1989) obtained by treatment of infected cells with the detergent nonidet P40 in presence of high ionic strength. Proteins were resolved on 17% acrylamide gels and transferred to nitrocellulose filters. Transfer conditions and subsequent analysis were the same as previously described (Alcaraz et a/., 1990).
Affinity
purification
of antibodies
267
from Western blots
Antibodies that bind to a given polypeptide band were purified from polyclonal sera by a simple modification of the Western blotting technique (Olmsted, 1981; Smith and Fisher, 1984). Cytoplasmic ASFV soluble antigen obtained as described (Escribano et a/., 1989) from 20-30 X lo6 infected cells, and 10” cpm of the same antigen labeled with [35S]methionine (Alcarazetal., 1990) were loaded into a large preparative well of a 17% acrylamide gel, electrophoretically separated, and then transferred to a nitrocellulose filter. The filter was dried and exposed to X-ray film to localize the ASFV-induced proteins. After protein localization, areas of the blot that contained the antigen were excised using a sharp scalpel. For affinity purification of antibodies, strips containing the different antigens were incubated with a hyperimmune serum after blocking with 2% nonfat dry milk. The antiserum was used diluted at 1: 10 in washing buffer (2% nonfat dry milk, 0.05% Tween in PBS, pH 7.2). Strips were washed six times with washing buffer and monospecific antibodies were eluted with 0.1 M glycine, pH 2.8, for 15 min with agitation. The pH of the eluted antibodies was neutralized with 1:5 volume 0.1 M NaOH in 50 mM Tris-HCI, pH 7. The strips were extensively washed and used more than five times. Antibodies were dialyzed against distilled water, lyophiIized, and resuspended in about 1 :lO of the original volume.
lmmunofluorescence
and flow cytometric
analysis
Separate ASFV-infected MS cells at different hpi obtained after incubation with 10 mM EDTA in PBS, pH 7.2, at 4” were washed with staining buffer. Cells were then incubated with either hyperimmune serum at 1:40 dilution or nondiluted monospecific antiserum (previously determined as saturating antibody concentrations) for 30 min at 4”. Then, cells were washed twice with staining buffer and incubated with a second FITC rabbit anti-swine IgG at 1:30 dilution for 30 min at 4”. After two more washes with staining buffer, cells were fixed with 1% paraformaldehyde in staining buffer. Stained cells were run in a FACStar PLUS (Becton Dickinson, San Jose, CA) flow cytometer equipped with a 2-watt argon laser tuned to 488 nm. Data were analyzed with FACStar PLUS Research Software (BectonDickinson) in a C30 computer. LYSYS II C32 Software (Becton-Dickinson) was used to obtain Fig. 1 B. Fluorescence data were collected by using logarithmic amplification on 5000 cells as determined by forward light scatter intensity. One-color fluorescence data were displayed as immunofluorescence profiles in which log
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FSCFIG. 1. Expression of ASFV-specified antigens on the plasma membranes of infected cells. (A) Flow cytometric analysis of ASFV antigen expression. x-axis represents the size of sample (FSC), whereas the y-axis represents the relative fluorescence intensity (log-scale) of the cell population at 0 hpi (Oh) and 6 hpi (6h). (B) Flow cytometric analysis of the relative cell number that presents different fluorescence intensity at 0, 6, 12, and 20 hpi.
fluorescence intensity was plotted on the x axis and cell number on they axis.
RESULTS Cell surface expression of ASFV-induced plasma membrane proteins By flow cytometric analysis of unfixed cells incubated with hyperimmune serum at 0, 6, 12, and 20 hpi over 80% of infected cells showed specific fluorescence at 6 hpi (Fig. 1A). This indicated that one or more ASFV antigens were expressed on the cell surface at this time point. Infected cells increased their relative fluorescence at 12 and 20 hpi with respect to that obtained at 6 hpi (Fig. 1 B). No significant differences in the percentage of cells that expressed viral antigens on their surfaces-over 90%-or their relative fluorescence intensities were found between 12 and 20 hpi. This result suggested maximum viral antigen expression at about 12 hpi.
Kinetics of individual protein expression on the cell surface Affinity-purified monospecific antisera against six virus-induced plasma membrane proteins with relative molecular weights of 12K, 15K, 16K, 23.5K, 30K, and 35K, and another against the major viral structural protein of 73K, were obtained from a recovered pig serum after ASFV infection as described in materials and methods. All antisera reacted with only one protein by Western blotting, with the exception of the antiserum raised against the 35K polypeptide, which also reacted
AND ESCRIBANO
with a higher molecular weight polypeptide of about 60K, probably a precursor protein (Fig. 2). Seven antisera were used in a flow cytometric analysis of infected cells at 0, 6, 12, and 20 hpi. Each antiserum reacted with infected unfixed cells as early as 6 hpi with the exception of antiserum against ~35, which first gave significant fluorescence at 12 hpi, and antiserum against ~73, which did not react at any time after infection (Fig. 3). The percentage of infected cells that expressed each protein at different hpi is shown in Fig. 4. Protein pl2 was detected on the cell surface in at least 91% of infected cells. Polypeptides ~15, ~16, ~23.5, ~30, and p35 were expressed in 78, 85, 83, 82, and 78% of infected cells, respectively. The maximum percentage of infected cells that expressed proteins ~12, ~15, ~16, ~23.5, and p30 was obtained at 6 hpi and did not vary significantly at 12 and 20 hpi. Antiserum against p35 reacted with the maximum percentage of unfixed infected cells from 12 hpi (Fig. 4).
Immune response against virus-induced membrane proteins
plasma
Pigs 1, 2, and 3, orally inoculated with attenuated ASFV E,, CVl-4 strain, and pig 4 which survived intra-
I2345678
-73K
-35 K -3OK -23.5
K
-16K -15K -12K FIG. 2. Affinity purification of antibodies against seven virus-induced proteins. Antibodies against the virus-specified proteins p73 (lane l), p35 (lane 2) p30 (lane 3) ~23.5 (lane 4) pl6 (lane 5) pl5 (lane 6) and pl2 (lane 7) were affinity purified from an hyperimmune serum. Their specificity was characterized by Western blotting on nitrocellulose strips containing a soluble antigen obtained from infected cells. The hyperimmune serum used in the affinity purification of monospecific antibodies was used as control in Western blotting (lane 8).
AFRICAN
SWINE FEVER-INDUCED
PI2
P23.5
1
P30
P73
I
I
-
Rd0tive
fluorescence
intensity -
FIG. 3. Krnetrc expression of rndrvrdual ASFV-specified plasma membrane proterns. These are fluorescence histograms that result from flow cytometric analyses of unfixed ASFV-infected cells at 0 hpr (l), 6 hpi (2) 12 hpr (3) and 20 hpr (4) incubated with six monospecific antisera against viral plasma membrane proterns (~12, ~15, ~16, ~23.5, ~30, and ~35) plus a FITC rabbit anti-swine serum. The serum against viral protein p73 was used as a representatrve control of internal nonexpressed protein on the surface of Infected cells, Fluorescence Intensity is presented (log-scale) on the x-axis, whereas the y-axis represents the relative number of cells.
muscular inoculation of the same virus; were used to study antibody induction against viral plasma membrane proteins. Pigs 1, 2, and 3 had an inapparent disease and shorter viraemias, 15-20 days, than pig 4, which showed appreciable clinical signs and 30 days of viraemia (data not shown). Moreover, the virus titers in peripheral blood were at least four logarithms higher in pig 4 than in pigs 1, 2, and 3 (data not shown). These differences in viraemias and clinical signs, depending on the inoculation method used, probably resulted in pig 4 presenting higher specific antibody titer at 40 dpi than pigs 1, 2, and 3 (Figs. 5A and 7A). All pigs were bled before inoculation and on Postinoculation Days 5, 11, 20, and 40. Antibodies against virus-induced plasma membrane proteins were detected from Postinoculation Day 1 1 by Western blotting in all pigs. Antibodies against ~12, ~23.5, and p30 were detected on Day 11 postinfection. At 20 dpi, antibodies against all
PLASMA
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PROTEINS
the previously characterized plasma membrane proteins were detected in pigs 1, 2, and 3, but pig 4 needed 40 days to induce antibodies against protein p14 (Fig. 5A; Fig. 7B). Interestingly, protein pl2 was less reactive with antibodies obtained at Days 20 and 40 after infection than those obtained at Day 1 1 in pig 3 (data not shown) and in Pig 4 (Fig. 7B), but not in pig 1 (Fig. 5A). These sera were also used in flow cytometric analysis of cells at 0, 6, 12, and 20 hpi. We found specific fluorescence in unfixed infected cells using sera from Days 1 1 to 40 after infection in all inoculated pigs (Figs. 5B, 6, 7A). However, early viral proteins, expressed on the cell surfaces before newly formed infective virus particles could be released from infected cells after potential antibody-mediated ceil lysis, reacted significantly only with sera obtained after 20 dpi. Sera obtained from both orally and intramuscularly inoculated pigs at 20 and 40 dpi recognized similar percentages of infected cells and also showed similar behavior relative to the time after infection when antibodies recognized infected cells (Figs. 6, 7A). Apparently, antibodies to viral epitopes exposed on the surface of infected cells reached a plateau at about 20 dpi In all inoculated pigs, even if the specific antibody titer against the virus was increased at later dpi, as shown in pig 4 (Fig. 7A).
DISCUSSION Virus-induced plasma membrane proteins can mediate in vitro the interaction of infected cells with cell-mediated and humoral components of the immune re1001
0 Hours
6
I2 after
20 infection
FIG. 4. Percentage of ASFV-Infected cells expressing different viral anttgens on the cell surface. This figure, based on the flow cytometry results shown in Fig. 3, represents the percentage of fluorescent cells obtained at 0, 6, 12, and 20 hpr after incubation with the monospecific antisera against vrral specrfied proterns pl2 (A), pl5 (+), pl6 (m), ~23.5 (A), p30 (II), p35 (0) and p73 (0) plus FITC rabbrt antrswine serum. Data are expressed as the arithmetic mean of three fluorescence assays carried out with each monospecifrc serum; the standard deviatron was lower than 4% in every case.
270
ALCARAZ,
ALVAREZ,
AND ESCRIBANO
B dpi ----P 0
9.6.T--
5
11
20
40
.zii 6.. 8m 6.- 4-3-2 -I --
Days after infection
Raktive
cell number
FIG. 5. Antibody response to ASFV-specified and plasma membrane proteins in orally inoculated prgs. (A) (I) Analysis of the specific antibody ELISA titer in three ASFV orally inoculated pigs numbered as 1 (0) 2 (m), and 3 (A) measured by ELISA titer obtained at different dpi. (II) Western blotting reactions on nitrocellulose strips containing soluble cytoplasmic antigen from infected cells with sera obtained from pig 1 before inoculation and at Postinoculation Days 5, 11, 20, and 40. (B) Antibody response to virus-specified plasma membrane proteins in pig 1, representative of the orally inoculated pigs. The relative number of fluorescent cells was analyzed by flow cytometry at 0, 6, 12, and 20 hpi when incubated with pig sera obtained before inoculation (0) and at 5, 11, 20, and 40 dpi plus FITC rabbit anti-swine serum. In fluorescence histograms, fluorescence intensity is represented (log-scale) on the x-axis, whereas the y-axis represents the relative number of cells.
sponse elicited during viral infections (Mallon et al., 1985; Goudsmit et al., 1988; Falgout et al., 1990; Laviada eta/., 1990; Schlesinger et al., 1990). If an in viva correlate of this phenomenon can be established, the virus specified cell-surface antigens could play an important role during the immune response in ASFV-infected pigs.
I
I
0
6 I2 Hours after infection
20
FIG.6. Average percentage of ASFV-infected cells expressing viral antigens on the cell surface which react with sera obtained from the three orally infected pigs at different days after infection. This figure shows the percentage of fluorescent ASFV-infected cells at 0, 6, 12, and 20 hpi after reaction with sera from the infected pigs obtained at 0 (0) 5 (m), 11 (A), 20 (Cl), and 40 dpi (A) plus FITC rabbit anti-swine serum.
The results of our experiments confirm and extend the observation that ASFV antigens are expressed on the plasma membrane of infected cells. The data provide evidence that the majority of viral antigens on infected cell surfaces are expressed at early times after infection, before newly infective virus is produced. Previous studies have indicated that all proteins detected on the cell surface are synthesized in the presence of phosphonoacetic acid, which only permits synthesis of proteins before viral DNA replication (Alcaraz et al., 1989). However, in this study, p35 was expressed in large quantities later than the other antigens. Evidence for a 32K polypeptide that appears later in virus-infected Vero cell membranes than other viral antigens has been previously described (Santa&n and Vifiuela, 1986). In viruses with similar genomic organization to ASFV, such as vaccinia virus, about nine virus-induced proteins are expressed on the surface of infected cells (Mallon and Holowczak, 1986). The orthopoxvirusspecified antigens that become associated with the plasma membrane of infected cells can be divided into three categories (Oie and Ichihashi, 1981). Two of these are the “early” viral antigens expressed prior to the onset of viral DNA synthesis (Ueda et al., 1969, 1972; Koszinowski et al., 1976) and the “late” viral antigens which are expressed during or after viral DNA replication is completed (Weintraub and Dales, 1974; Dales and Oldstone, 1982). The third group consists of
AFRICAN
SWINE FEVER-INDUCED
PLASMA
MEMBRANE
PROTEINS
0 5 II
271
2040
= I-73,
Days after infection
w
. ,*
--l6K B-l5K
_
=i-l2K
Hours after infection FIG. 7. Antibody response to ASFV-specified and plasma membrane proterns in a rntramuscularly Inoculated prg. (A) Comparison of the antibody titer in ELISA (top) and vrral plasma membrane recognition at 0, 6, 12, and 20 hpi by sera obtained at 5 (0) 1 1 (m), 20 (e), and 40 (A) dpt by flow cytometry (bottom). (B) Western blotting reactions on nitrocellulose strips contarnrng soluble cytoplasmic ASFV antigens from infected cells with sera obtained from pig 4 intramuscularly inoculated at time before inoculation (lane 0) and at 5 (lane 5), 1 1 (lane 1 l), 20 (lane 20), and 40 dpr (lane 40).
virion antigens transferred to the plasma membrane as a result of fusion of the viral envelope with the plasma membrane during adsorption and penetration of virus particles into cells (Oie and Ichihashi, 1981). This last category of ASFV proteins was not studied in the present work. Proteins ~12, ~15, ~16, ~23.5, and p30 can be classified as “early” expressed proteins and protein p35 as a “late” viral plasma membrane antigen. Complement-dependent antibody-mediated cytotoxicity (CDAC) (Norley and Wardley, 1982) and antibodydependent cell-mediated cytotoxicity (ADCC) (Wardley et a/., 1985) have been described in African swine fever. These immune mechanisms can be elicited by one or more viral plasma membrane proteins (Alcaraz et al., 1989). Antibodies produced during ASFV infection partially protected pigs that were intraperitoneally passively immunized (Schlaferetal., 1984b; Wardleyet al., 1985) or suckling piglets (Schlafer et al., 1984a,b) against the effects of virulent ASFV challenge. Protection, which was exemplified by a reduction in pyrexia and viraemia plus an increased survival time, appeared to be mediated through the effects of CDAC and ADCC immune mechanisms (Wardley et al., 1985). These authors suggested that reduction in viraemia was associated with complement lysis, whereas protection was conferred by ADCC. Effective lysis of infected cells mediated by antibodies reacting with viral plasma membrane proteins is determined by two factors: (i) viral antigen concentration on the cell surface and (ii) titer and isotype of the
antibodies reacting with the viral antigens. For most virus-induced plasma membrane proteins, the quantity of antigen expressed on the surface of infected cells reached a plateau between 6 and 12 hpi. By using sera from infected pigs obtained at 20 and 40 dpi, the fluorescence intensities and percentages of positive cells at 6, 12, and 20 hpi were very similar. This indicates that viral antigen concentration sufficient to elicit lysis of infected cells mediated by antibodies could be reached very soon after infection, prior to formation of new infective viral particles. Development of antibody capable of initiating CDAC and ADCC has been described as a relatively late event in infected pigs (Norley and Wardley, 1982). This supports our results obtained after analysis of the antibody response against virus-induced plasma membrane proteins. We have shown that infected pigs need about 20 days after infection to develop optimal recognition of viral antigens expressed on the cell surfaces at early times after infection. However, viral plasma membrane proteins such as pl2 and p30 induced a considerable amount of antibody, detected by Western blotting, as early as 11 dpi and could play an important role in the lysis of infected cells in the first stages of the disease. No experiments have been carried out in naive pigs to demonstrate the protective efficiency in viva after transfer of antibodies obtained at different times after infection. However, in vitro, antibodies obtained at 1415 dpi but not at 5-7 dpi were effective in fixing complement to a degree sufficient for cell lysis (Norley and
272
ALCARAZ,
ALVAREZ,
Wardley, 1982). At 14-l 5 dpi the same authors obtained the maximum percentage of complement-mediated lysis, suggesting that the onset of this mechanism is not simply the result of increasing levels of specific antibody in the serum. We found that antibodies in sera obtained at 20 and 40 dpi are the most efficient with respect to the relative number of infected cells they recognize and the fluorescence intensity they provide to the cells at 6 hpi. African swine fever virus does not produce easily detectable neutralizing antibodies. However, since there is evidence of ASFV challenge protection by antibodies, we have focused attention on structural and nonstructural ASFV-induced proteins which are expressed on the surface of infected cells. Recent studies with flaviviruses have demonstrated that protective immunity can be elicited by a nonstructural protein, NSl, which is expressed on the cell surface (Falgout et a/., 1990; Putnak and Schlessinger, 1990). Similar results were obtained with plasma membrane proteins in other viral diseases, such as mouse hepatitis virus type 3 (Leconte et a/., 1987). Experiments to elucidate the relevance of the characterized ASFV plasma membrane proteins in the immunological mechanisms involved in protection against this disease are underway. ACKNOWLEDGMENTS This work was supported by a grant from the lnstituto National de Investigaci6n y Tecnologfa Agraria y Alimentaria (INIA). We thank Doctors D.L. Rock, D.P. Schenkein, F. Ruiz-Gonzalvo, and M. De Diego for useful discussions.
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