INFECTION AND IMMUNITy, Apr. 1975, p. 829-834 Copyright i 1975 American Society for Microbiology
Vol. 11, No. 4 Printed in U.S.A.
Comparative Studies of Visna and Maedi Viruses
as
Antigens
P. D. MEHTA* AND H. THORMAR New York State Institute for Basic Research in Mental Retardation, Staten Island, New York 10314 Received for publication 8 November 1974
Rabbits were immunized with purified visna and maedi viruses, using complete Freund adjuvant, in footpad and intramuscular sites. The resulting antisera and their isolated immunoglobulin G (IgG) and M (IgM) classes were evaluated by tanned-cell passive hemagglutination (PHA), complement fixation, gel diffusion, and virus neutralization tests. Early, intermediate, and late bleedings showed increasingly high antibody activities by the PHA, complement fixation, and gel diffusion tests. The activities were associated mainly with the IgG class, although low, but significant activities were also found in the IgM class, as detected by PHA and complement fixation tests. Both antibody classes appeared at the same time during the course of immunization. The viruses, when tested against specific rabbit anti-visna and anti-maedi sera in gel diffusion tests, showed the presence of one to two precipitin lines depending upon the antibody concentration of the sera. A low amount of neutralizing activity was demonstrated in late bleedings but was not seen in earlier bleedings. The neutralizing activity against visna virus in immunized rabbit sera was found to be associated only with the IgG class. Visna and maedi viruses appeared to be immunologically identical when examined in gel diffusion tests and showed the same degree of inhibition when compared in passive hemagglutination inhibition tests using antisera made specific for each virus.
Visna and maedi viruses are closely related with respect to morphological and biological properties (13). Furthermore, a partial serological cross-reaction between visna and maedi viruses has been shown by fluorescent antibody and virus neutralization techniques, using sera obtained from infected sheep (14, 15). Although a number of studies have been reported on sheep infected with visna and maedi viruses, little work has been done on rabbits immunized with these viruses. Recently, Karl and Thormar (3) immunized rabbits with visna virus and demonstrated that antibody activity to the virus was present in immunoglobulin G (IgG) and immunoglobulin M (IgM) fractions of hyperimmunized sera. The object of this investigation was to study hemagglutinating, complement fixing, neutralizing, and' precipitating antibody activities in isolated IgG and IgM classes in sera obtained at intervals from rabbits after immunization with visna and maedi viruses. In addition, these viruses were compared immunologically with each other, using rabbit antisera made specific for the viruses.
were grown in 250-ml Falcon plastic flasks and maintained in Eagle basal medium with 0.2% bovine serum albumin, as described previously (5). Virus purification. Visna virus, strain K796, and maedi virus, strain M88, were used. Infectious tissue culture fluid was harvested, and the virus was concentrated and purified by density gradient centrifugation (6). The titer of the purified virus preparations used in the present studies was 106 median tissue culture infective doses (TCID.0) per ml. Preparation of antisera. Antisera to purified visna and maedi viruses were prepared in rabbits by injecting 0.5 mg of antigen, first with complete Freund adjuvant into the footpads and then intramuscularly. The rabbits were bled 3 weeks after the initial injection and 1 to 2 weeks after each subsequent
MATERIALS AND METHODS Cell cultures and media. Cultures of sheep choroid plexus cells were used for production of virus. They 829
injection. The rabbit antisera were made specific for virus antigen by suitable absorption with lyophilized preparations of sheep serum, sheep liver, and bovine serum albumin. The mixture was incubated at 37 C for 1 h, refrigerated overnight, and centrifuged at 12,000 rpm for 15 min. The absorbed sera did not show any precipitin line in the Ouchterlony analysis (8) when tested against sheep serum, sheep liver, and bovine serum albumin. The absorbed sera were further tested in the Ouchterlony test against tissue culture fluid from uninfected sheep cells to rule out the presence of a contaminating antigen. The tissue culture fluid preparation did show precipitin lines against unabsorbed rabbit anti-visna and anti-maedi
830
INFECT. IMMUN.
MEHTA AND THORMAR
but failed to show any precipitin reaction against absorbed sera. PHA. This passive hemagglutination (PHA) test was carried out according to the method of Stavitsky (11). Sheep erythrocytes were treated with tannic acid and coated with purified virus. In initial tests, maximum serum titers were obtained with erythrocytes coated with a 120 ug/ml solution of the virus. This virus dilution was therefore used as the standard coating level in subsequent tests. All antisera were inactivated at 56 C for 30 min. Readings were made after 2 to 3 h at room temperature and after incubation overnight at 4 C. In the PHA inhibition test, twofold dilutions of each purified virus preparation to be used as inhibitor were made in test tubes in 0.25-ml quantities. An equal volume of antiserum, diluted to a concentration that was two to four times its usual 1-plus end point concentration in the PHA test, was added to each tube, and the mixture was incubated at room temperature for 1 h. Tanned cells (0.05 ml) coated with the respective virus were added to each tube and read as described earlier. CF test. The complement fixation (CF) test was carried out by the microtiter technique described by Sever (10) with purified virus as antigen. In the procedure, 0.025 ml of viral antigen (106) TCID60 per ml and protein content of 120 jug/ml), 0.025 ml of specific antiserum or antibody fractions, and 0.05 ml of complement (2 units) were mixed and incubated overnight at 4 C. After incubation, 0.05 ml of sensitized sheep erythrocytes was added and the mixture was incubated at 37 C for 30 min. CF titers were determined under conditions where no anti-complementary activity could be demonstrated. Immunodiffusion and immunoelectrophoresis. Double diffusion in agar gel was performed by the method of Ouchterlony (8) and immunoelectrophoresis on glass microscopic slides was employed as described by Scheidegger (9). Quantitation of IgG and IgM. IgG and IgM concentrations in pre- and post-immunized sera were determined by the radial immunodiffusion method (1) in agar, using guinea pig antisera to rabbit IgG and IgM.
sera
Neutralization test. A virus neutralization test carried out as described previously (15). Serial twofold dilutions of serum or isolated antibody fractions were mixed with an equal volume of a virus dilution containing approximately 200 TCID50 per 0.1 ml. The mixtures were incubated overnight at 22 C and were then inoculated with 0.1-ml amounts into tissue culture tubes. The tubes were incubated for 14 days before final reading. The neutralizing activities of the isolated immunoglobulin fractions are expressed in titers per 0.1 ml of protein adjusted to the concentration (milligrams per milliliter) found in the was
serum.
Isolation of IgG and IgM. Approximately 15 ml of serum was centrifuged at 33,000 rpm in the Spinco model L-50 ultracentrifuge for 17 h at 4 C. The pellet was mixed with a few drops of the fluid above it, and the mixture was subjected to zone electrophoresis on starch (4) for 16 to 18 h at a potential gradient of 20 V/inch, using barbital buffer (pH 8.6) and ionic strength of 0.05. Each 0.5-inch (about 1.25-cm) portion of the block was eluted with 0.15 M NaCl, concentrated, and tested in immunoelectrophoresis with potent goat anti-rabbit serum. The separation of IgM from IgG was further achieved by gel filtration of selected starch block eluates on Sephadex G-200 column, equilibrated with 0.1 M tris(hydroxymethyl)aminomethane-0.15 M NaCl, pH 8.0. Determination of protein. Protein determination of viral antigens was performed by the method of Lowry et al. (7) with bovine serum albumin as standard. The protein concentrations of the immunoglobulins were determined by using an extinction coefficient [EAcnm 1cm] of 12 for IgM and 14 for IgG. RESULTS
Production of antibodies against visna and maedi viruses in rabbits. The immunization and bleeding schedule of rabbits injected with visna virus is shown in Table 1. After suitable absorption as described above, the entire series of bleedings from each rabbit was tested by PHA, CF, gel diffusion, and neutralization tests against visna virus.
TABLE 1. Schedule of injections and bleedings of rabbits and PHA, CF, and neutralizing (N) titers of sera as tested against visna virus A91a
A9O0
Day 0 25 37 52 73 91 99 112 127 146 a
Injection
PHA
CF
N
200
256
1,600
A92a
PHA
CF
N
Vol. 11, No. 4 Printed in U.S.A.
Comparative Studies of Visna and Maedi Viruses
as
Antigens
P. D. MEHTA* AND H. THORMAR New York State Institute for Basic Research in Mental Retardation, Staten Island, New York 10314 Received for publication 8 November 1974
Rabbits were immunized with purified visna and maedi viruses, using complete Freund adjuvant, in footpad and intramuscular sites. The resulting antisera and their isolated immunoglobulin G (IgG) and M (IgM) classes were evaluated by tanned-cell passive hemagglutination (PHA), complement fixation, gel diffusion, and virus neutralization tests. Early, intermediate, and late bleedings showed increasingly high antibody activities by the PHA, complement fixation, and gel diffusion tests. The activities were associated mainly with the IgG class, although low, but significant activities were also found in the IgM class, as detected by PHA and complement fixation tests. Both antibody classes appeared at the same time during the course of immunization. The viruses, when tested against specific rabbit anti-visna and anti-maedi sera in gel diffusion tests, showed the presence of one to two precipitin lines depending upon the antibody concentration of the sera. A low amount of neutralizing activity was demonstrated in late bleedings but was not seen in earlier bleedings. The neutralizing activity against visna virus in immunized rabbit sera was found to be associated only with the IgG class. Visna and maedi viruses appeared to be immunologically identical when examined in gel diffusion tests and showed the same degree of inhibition when compared in passive hemagglutination inhibition tests using antisera made specific for each virus.
Visna and maedi viruses are closely related with respect to morphological and biological properties (13). Furthermore, a partial serological cross-reaction between visna and maedi viruses has been shown by fluorescent antibody and virus neutralization techniques, using sera obtained from infected sheep (14, 15). Although a number of studies have been reported on sheep infected with visna and maedi viruses, little work has been done on rabbits immunized with these viruses. Recently, Karl and Thormar (3) immunized rabbits with visna virus and demonstrated that antibody activity to the virus was present in immunoglobulin G (IgG) and immunoglobulin M (IgM) fractions of hyperimmunized sera. The object of this investigation was to study hemagglutinating, complement fixing, neutralizing, and' precipitating antibody activities in isolated IgG and IgM classes in sera obtained at intervals from rabbits after immunization with visna and maedi viruses. In addition, these viruses were compared immunologically with each other, using rabbit antisera made specific for the viruses.
were grown in 250-ml Falcon plastic flasks and maintained in Eagle basal medium with 0.2% bovine serum albumin, as described previously (5). Virus purification. Visna virus, strain K796, and maedi virus, strain M88, were used. Infectious tissue culture fluid was harvested, and the virus was concentrated and purified by density gradient centrifugation (6). The titer of the purified virus preparations used in the present studies was 106 median tissue culture infective doses (TCID.0) per ml. Preparation of antisera. Antisera to purified visna and maedi viruses were prepared in rabbits by injecting 0.5 mg of antigen, first with complete Freund adjuvant into the footpads and then intramuscularly. The rabbits were bled 3 weeks after the initial injection and 1 to 2 weeks after each subsequent
MATERIALS AND METHODS Cell cultures and media. Cultures of sheep choroid plexus cells were used for production of virus. They 829
injection. The rabbit antisera were made specific for virus antigen by suitable absorption with lyophilized preparations of sheep serum, sheep liver, and bovine serum albumin. The mixture was incubated at 37 C for 1 h, refrigerated overnight, and centrifuged at 12,000 rpm for 15 min. The absorbed sera did not show any precipitin line in the Ouchterlony analysis (8) when tested against sheep serum, sheep liver, and bovine serum albumin. The absorbed sera were further tested in the Ouchterlony test against tissue culture fluid from uninfected sheep cells to rule out the presence of a contaminating antigen. The tissue culture fluid preparation did show precipitin lines against unabsorbed rabbit anti-visna and anti-maedi
830
INFECT. IMMUN.
MEHTA AND THORMAR
but failed to show any precipitin reaction against absorbed sera. PHA. This passive hemagglutination (PHA) test was carried out according to the method of Stavitsky (11). Sheep erythrocytes were treated with tannic acid and coated with purified virus. In initial tests, maximum serum titers were obtained with erythrocytes coated with a 120 ug/ml solution of the virus. This virus dilution was therefore used as the standard coating level in subsequent tests. All antisera were inactivated at 56 C for 30 min. Readings were made after 2 to 3 h at room temperature and after incubation overnight at 4 C. In the PHA inhibition test, twofold dilutions of each purified virus preparation to be used as inhibitor were made in test tubes in 0.25-ml quantities. An equal volume of antiserum, diluted to a concentration that was two to four times its usual 1-plus end point concentration in the PHA test, was added to each tube, and the mixture was incubated at room temperature for 1 h. Tanned cells (0.05 ml) coated with the respective virus were added to each tube and read as described earlier. CF test. The complement fixation (CF) test was carried out by the microtiter technique described by Sever (10) with purified virus as antigen. In the procedure, 0.025 ml of viral antigen (106) TCID60 per ml and protein content of 120 jug/ml), 0.025 ml of specific antiserum or antibody fractions, and 0.05 ml of complement (2 units) were mixed and incubated overnight at 4 C. After incubation, 0.05 ml of sensitized sheep erythrocytes was added and the mixture was incubated at 37 C for 30 min. CF titers were determined under conditions where no anti-complementary activity could be demonstrated. Immunodiffusion and immunoelectrophoresis. Double diffusion in agar gel was performed by the method of Ouchterlony (8) and immunoelectrophoresis on glass microscopic slides was employed as described by Scheidegger (9). Quantitation of IgG and IgM. IgG and IgM concentrations in pre- and post-immunized sera were determined by the radial immunodiffusion method (1) in agar, using guinea pig antisera to rabbit IgG and IgM.
sera
Neutralization test. A virus neutralization test carried out as described previously (15). Serial twofold dilutions of serum or isolated antibody fractions were mixed with an equal volume of a virus dilution containing approximately 200 TCID50 per 0.1 ml. The mixtures were incubated overnight at 22 C and were then inoculated with 0.1-ml amounts into tissue culture tubes. The tubes were incubated for 14 days before final reading. The neutralizing activities of the isolated immunoglobulin fractions are expressed in titers per 0.1 ml of protein adjusted to the concentration (milligrams per milliliter) found in the was
serum.
Isolation of IgG and IgM. Approximately 15 ml of serum was centrifuged at 33,000 rpm in the Spinco model L-50 ultracentrifuge for 17 h at 4 C. The pellet was mixed with a few drops of the fluid above it, and the mixture was subjected to zone electrophoresis on starch (4) for 16 to 18 h at a potential gradient of 20 V/inch, using barbital buffer (pH 8.6) and ionic strength of 0.05. Each 0.5-inch (about 1.25-cm) portion of the block was eluted with 0.15 M NaCl, concentrated, and tested in immunoelectrophoresis with potent goat anti-rabbit serum. The separation of IgM from IgG was further achieved by gel filtration of selected starch block eluates on Sephadex G-200 column, equilibrated with 0.1 M tris(hydroxymethyl)aminomethane-0.15 M NaCl, pH 8.0. Determination of protein. Protein determination of viral antigens was performed by the method of Lowry et al. (7) with bovine serum albumin as standard. The protein concentrations of the immunoglobulins were determined by using an extinction coefficient [EAcnm 1cm] of 12 for IgM and 14 for IgG. RESULTS
Production of antibodies against visna and maedi viruses in rabbits. The immunization and bleeding schedule of rabbits injected with visna virus is shown in Table 1. After suitable absorption as described above, the entire series of bleedings from each rabbit was tested by PHA, CF, gel diffusion, and neutralization tests against visna virus.
TABLE 1. Schedule of injections and bleedings of rabbits and PHA, CF, and neutralizing (N) titers of sera as tested against visna virus A91a
A9O0
Day 0 25 37 52 73 91 99 112 127 146 a
Injection
PHA
CF
N
200
256
1,600
A92a
PHA
CF
N
