Immunology 1979 38 765
Immune mechanisms against canine distemper II.
ROLE OF ANTIBODY IN ANTIGEN MODULATION AND PREVENTION OF
INTERCELLULAR AND EXTRACELLULAR SPREAD OF CANINE DISTEMPER VIRUS
C. K. HO & L. A. BABIUK Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada
Acceptedfor publication 19 July 1979
Summary. Specific antibody was shown to be highly effective in neutralizing extracellular canine distemper virus (CDV) as well as preventing the intercellular spread of this virus. Thus, relatively low levels of antibody neutralized 1 x 105 TCID50 of extracellular CDV and the development of plaques or CPE in Hep-2 and Vero cells respectively could be prevented even when up to 5% of the cells were infected. This inhibition of CPE and virus spread was most pronounced when antibody was added early but could still limit the degree of CPE if added as late as 48 h post-infection. This anti-viral activity was observed in different cell types including canine macrophages, cells normally infected with CDV in vivo. Prolonged exposure of infected target cells to high concentrations of antibody led to redistribution of surface viral antigens and their subsequent disappearance. The possible role of antibody in the defence against, and/or recovery from CDV and the mechanism(s) by which antibody may aid in recovery are discussed.
despite the availability of effective vaccines. Evidence from both experimental and natural cases of canine distemper shows that the outcome of infection depends on the ability of the animal to develop an effective immune response. Thus, if the level of neutralizing antibody of an infected dog reaches a protective level within 2 weeks after exposure to canine distemper virus (CDV) the outcome of the infection is usually mild and there is rapid viral clearance from the lymphatic tissues of the animal (Appel, 1969; 1970). Conversely, the lack of an early progressive response is associated with fatal distemper infection, possibly due to extensive virus replication in lymphoid tissues and subsequent immunosuppression. Several immune mechanisms have been proposed as responsible for recovery or for providing protection against CDV either as a result of previous natural exposure or through vaccination. The first of these mechanisms is the 'blocking' of CDV replication by attenuated CDV or measles viruses either at the site of entry or in other target organs. This blockage is not fully understood but it has been postulated that it may arise from the production of either defective interfering virus particles or due to interferon (Epstein, Stevens & Merigan, 1972; Huang, 1973, Lodmell & Notkins, 1974). Secondly, cell-mediated immunity, although not studied extensively in canine distemper may be important in killing virus infected cells prior to the release of large quantities of virus and infection of vital target organs (Appel, 1970). Finally, antibody levels have definitely been shown to correlate with the
INTRODUCTION Canine distemper is a major hazard in canine medicine Correspondence: Dr L. A. Babiuk, Department ofVeterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO.
0019-2805/79/1200-0765$02.00 © 1979 Blackwell Scientific Publications
765
C. K. Ho & L. A. Babiuk
766
eventual outcome of the disease (Appel, 1970; Liv & Coffin, 1957; Rockborn, 1958). Whether antibody is involved exclusively in neutralizing extracellular virus and thus preventing re-infection or whether it also plays a role in removing the virus coded antigens on infected cells (Joseph & Oldstone, 1975) with subsequent prevention of viral spread via the intercellular route has not been elucidated (Notkins, 1974). The present investigation was designed to determine how well anti-CDV antiserum can neutralize extracellular viruses and whether antibody (Ab) can prevent the intercellular spread of CDV in vitro. MATERIALS AND METHODS Cells Vero and Hep-2 cells (a human carcinoma cell line) were grown in 100 mm plastic tissue culture plates (Corning No. 25020) in Eagle's minimum essential medium (MEM) supplemented with 10% heat-inactivated (56° for 30 min) foetal calf serum (FCS), 0 1 mm glutamine, I1% non-essential animo acids and 50 ,g/ml of gentamycin. The cells were incubated at 37° in a humidified incubator with 5% CO2 and were maintained at a confluent or subconfluent level by serial passages.
Virus The green strain of canine distemper virus (CDV) was propagated in Vero (African green monkey kidney) cells as described elsewhere (Ho & Babiuk, 1979b). Subconfluent monolayers of cells were infected at a multiplicity of infection (MOI) of 1 and 48 h later when extensive cytopathic degeneration was evident, the supernatant fluids were harvested and stored at 70° until use. The average titre of the virus was around 1 x 106 PFU/ml. -
Sera Control serum and anti-CDV antisera were kindly provided by Dr R. Schultz (Cornell University, Ithaca, N.Y.). All sera were from specific pathogen-free dogs, were heat-inactivated at 560 for 30 min and adsorbed twice with 1/5 volume of packed Vero or Hep-2 cells before use. Adsorption was done at 40 for 1 h in each instance. Neutralization of extracellular virus Subconfluent monolayers of vero and Hep-2 cells were cultured in microtitre plates (Falcon Plastic No. 3040).
Medium from each well was aspirated and 0 1 ml of diluted dog anti-CDV serum (100 TCID50 neutralizing units) or normal serum (no detectable CDV antibody) was added to each well. Simultaneously, 01 ml aliquots of various dilutions of CDV was added to each well and the entire contents were incubated at 350 in a humidified 5% CO2 incubator. Parallel experiments were conducted in which the concentration of antibody was varied but the quantity of virus remained constant ( 100 PFU). The kinetics of neutralization were determined by mixing 104 PFU of CDV with a standard amount of anti-CDV serum (100 TCID50 neutralizing units), the mixture was incubated for various periods of time at 25° and then added to Vero cells in microtitre plates. The extent of cytopathology was determined 36-48 h later as described previously (Ho & Babiuk, 1979b).
Inhibition of intercellular spread of virus by antibody Subconfluent monolayers of Hep-2 or Vero cells were infected with CDV at a MOI of 2, incubated at 370 for 4 h prior to trypsinization and removal of the cells from the monolayers. The cells were then washed three times in Hanks's balanced salt solution (HBSS) and incubated for 1 h in MEM + anti-CDV antibody (final titre I/ 50) to neutralize any unadsorbed or uncoated extracellular viruses. The cells were washed three times in MEM + 5% FCS, counted and resuspended to the appropriate cell concentration(s) in MEM prior to cocultivation with Hep-2 cells as described previously (Ho & Babiuk, 1979b). In some experiments a constant amount of CDV antibody (100 TCID5o neutralizing units) was added to cultures containing varying numbers of infected cells while in others a fixed number of infected cells were cultured in different concentrations of antibody. Three days later the monolayers were fixed, stained, and the number of plaques counted. In parallel experiments, canine macrophages were prepared as described previously (Ho & Babiuk, 1 979a). Peripheral blood leucocytes were cultured in vitro. The monolayers were vigorously washed daily for 10 days to remove lymphocytes and nonmacrophage cell types to yield a culture containing >99% macrophages. These cells were then infected with CDV and incubated a further 24 h at 370 to inactivate extracellular virus before the addition of a Hep-2 or Vero cell overlay either in the presence or absence of various concentrations of anti-CDV antibody. The Hep-2 or Vero macrophage co-cultivated cultures were left undisturbed for 3 days before fixing, staining and counting the number of plaques present.
Immune mechanisms against canine distemper II
To determine the kinetics of inhibition of intercellular spread of virus, Hep-2 and Vero cells were infected at a MOI of 2, trypsinized after 4 h of incubation at 370 and cocultivated with Vero or Hep-2 cells. At various time intervals antibody was added to the cultures. Three days later the cultures were terminated and the number of plaques was determined.
Neutralization of cel/fusion factor Vero cells were grown in MEM + 5% FCS in 35 mm tissue culture dishes (Corning No. 25,000) and when the cells became confluent, the culture medium was removed and different volumes of fresh CDV suspension (prepared by serial passages in Hep-2 cells) and dog anti-CDV antiserum or control normal serum were added to each plate. The cells were incubated at 37° in 3% CO2 for 2 h and then trypsinized. Detached cells from triplicate plates were pooled, washed twice in HBSS and diluted with MEM + 5% FCS to 1/5 the original cell concentration. One ml aliquots of each cell suspension were seeded onto 35 mm culture dishes containing a 100 mm2 glass cover slip and the plates were incubated at 370 for about 2 h. When all the cells were attached to the glass surface, the cover slips were removed, the cells were stained with haematoxylineosin stain and the number of cells containing more than one nucleus were counted using a light microscope. Neutralization ofhaemolysin activity Fresh monkey red blood cells were washed 3 x in Alsever's solution and once in phosphate-buffered saline (PBS, 0 1 M Na2HPO4, 0 15 M NaCl, pH 7 2) by centrifugation at 1000 g for 10 min. Packed monkey red blood cells (MRBC) (0 5 ml) were resuspended in 2 ml of PBS and 0 2 ml of [51Cr]-sodium chromate (51Cr, New England Nuclear, Dorval, P.Q.). The cells were incubated at 370 for 90 min with occasional shaking. Following the labelling process, excess 51Cr was removed by repeated washings of the MRBC with PBS. The labelled cells were resuspended in PBS + 01 mm magnesium chloride to a 10% final concentration and 0-2 ml aliquots were dispensed into plastic tissue culture tubes (Falcon No. 2054) containing fresh CDV with or without various concentrations of dog antiserum, control serum or MEM + 5% FCS. The tubes were incubated in a 37° waterbath with constant shaking. After 4 h of incubation, MRBC in each tube were pelleted by centrifugation at I000g for 10 min and half the supernatant was harvested and radioactivity was
767
determined by a gamma spectrophotometer. All tests were performed in triplicate. Antibody-induced modulation of viral surface antigens Vero cells were infected with CDV as described above and when CPE became visible, the cells were removed from the culture dishes by brief exposure to 0.025% trypsin in 0 0I0% EDTA, washed twice in MEM + I0% FCS and resuspended to a concentration of I x 106 cells/ml in plastic tissue culture tubes (Falcon No. 2054). The diluent was foetal calf serum containing 2 mg/ml fluorescein-isothiocyanate (FITC) conjugated dog anti-CDV gammaglobulin. Staining patterns of the cells were observed by fluorescent microscopy after the cells were incubated at different temperatures for various periods of time. Non-infected Vero cells were set up in parallel to serve as negative controls. RESULTS Inhibition of CDV infectivity and virus spread by antibody Since we could easily neutralize CDV with antibody we attempted to determine whether the degree of neutralization was consistent regardless of the cell type used for the neutralization test. Hep-2 cells were chosen because they produced easily visible plaques and Vero cells produced rapid cytopathological changes without plaque formation. Figure 1 illustrates that both systems were useful for demonstrating the neutralizing activity of antibody on extracellular virus.
a
Vb
a-
9 JA
m
Immune mechanisms against canine distemper II.
ROLE OF ANTIBODY IN ANTIGEN MODULATION AND PREVENTION OF
INTERCELLULAR AND EXTRACELLULAR SPREAD OF CANINE DISTEMPER VIRUS
C. K. HO & L. A. BABIUK Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada
Acceptedfor publication 19 July 1979
Summary. Specific antibody was shown to be highly effective in neutralizing extracellular canine distemper virus (CDV) as well as preventing the intercellular spread of this virus. Thus, relatively low levels of antibody neutralized 1 x 105 TCID50 of extracellular CDV and the development of plaques or CPE in Hep-2 and Vero cells respectively could be prevented even when up to 5% of the cells were infected. This inhibition of CPE and virus spread was most pronounced when antibody was added early but could still limit the degree of CPE if added as late as 48 h post-infection. This anti-viral activity was observed in different cell types including canine macrophages, cells normally infected with CDV in vivo. Prolonged exposure of infected target cells to high concentrations of antibody led to redistribution of surface viral antigens and their subsequent disappearance. The possible role of antibody in the defence against, and/or recovery from CDV and the mechanism(s) by which antibody may aid in recovery are discussed.
despite the availability of effective vaccines. Evidence from both experimental and natural cases of canine distemper shows that the outcome of infection depends on the ability of the animal to develop an effective immune response. Thus, if the level of neutralizing antibody of an infected dog reaches a protective level within 2 weeks after exposure to canine distemper virus (CDV) the outcome of the infection is usually mild and there is rapid viral clearance from the lymphatic tissues of the animal (Appel, 1969; 1970). Conversely, the lack of an early progressive response is associated with fatal distemper infection, possibly due to extensive virus replication in lymphoid tissues and subsequent immunosuppression. Several immune mechanisms have been proposed as responsible for recovery or for providing protection against CDV either as a result of previous natural exposure or through vaccination. The first of these mechanisms is the 'blocking' of CDV replication by attenuated CDV or measles viruses either at the site of entry or in other target organs. This blockage is not fully understood but it has been postulated that it may arise from the production of either defective interfering virus particles or due to interferon (Epstein, Stevens & Merigan, 1972; Huang, 1973, Lodmell & Notkins, 1974). Secondly, cell-mediated immunity, although not studied extensively in canine distemper may be important in killing virus infected cells prior to the release of large quantities of virus and infection of vital target organs (Appel, 1970). Finally, antibody levels have definitely been shown to correlate with the
INTRODUCTION Canine distemper is a major hazard in canine medicine Correspondence: Dr L. A. Babiuk, Department ofVeterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO.
0019-2805/79/1200-0765$02.00 © 1979 Blackwell Scientific Publications
765
C. K. Ho & L. A. Babiuk
766
eventual outcome of the disease (Appel, 1970; Liv & Coffin, 1957; Rockborn, 1958). Whether antibody is involved exclusively in neutralizing extracellular virus and thus preventing re-infection or whether it also plays a role in removing the virus coded antigens on infected cells (Joseph & Oldstone, 1975) with subsequent prevention of viral spread via the intercellular route has not been elucidated (Notkins, 1974). The present investigation was designed to determine how well anti-CDV antiserum can neutralize extracellular viruses and whether antibody (Ab) can prevent the intercellular spread of CDV in vitro. MATERIALS AND METHODS Cells Vero and Hep-2 cells (a human carcinoma cell line) were grown in 100 mm plastic tissue culture plates (Corning No. 25020) in Eagle's minimum essential medium (MEM) supplemented with 10% heat-inactivated (56° for 30 min) foetal calf serum (FCS), 0 1 mm glutamine, I1% non-essential animo acids and 50 ,g/ml of gentamycin. The cells were incubated at 37° in a humidified incubator with 5% CO2 and were maintained at a confluent or subconfluent level by serial passages.
Virus The green strain of canine distemper virus (CDV) was propagated in Vero (African green monkey kidney) cells as described elsewhere (Ho & Babiuk, 1979b). Subconfluent monolayers of cells were infected at a multiplicity of infection (MOI) of 1 and 48 h later when extensive cytopathic degeneration was evident, the supernatant fluids were harvested and stored at 70° until use. The average titre of the virus was around 1 x 106 PFU/ml. -
Sera Control serum and anti-CDV antisera were kindly provided by Dr R. Schultz (Cornell University, Ithaca, N.Y.). All sera were from specific pathogen-free dogs, were heat-inactivated at 560 for 30 min and adsorbed twice with 1/5 volume of packed Vero or Hep-2 cells before use. Adsorption was done at 40 for 1 h in each instance. Neutralization of extracellular virus Subconfluent monolayers of vero and Hep-2 cells were cultured in microtitre plates (Falcon Plastic No. 3040).
Medium from each well was aspirated and 0 1 ml of diluted dog anti-CDV serum (100 TCID50 neutralizing units) or normal serum (no detectable CDV antibody) was added to each well. Simultaneously, 01 ml aliquots of various dilutions of CDV was added to each well and the entire contents were incubated at 350 in a humidified 5% CO2 incubator. Parallel experiments were conducted in which the concentration of antibody was varied but the quantity of virus remained constant ( 100 PFU). The kinetics of neutralization were determined by mixing 104 PFU of CDV with a standard amount of anti-CDV serum (100 TCID50 neutralizing units), the mixture was incubated for various periods of time at 25° and then added to Vero cells in microtitre plates. The extent of cytopathology was determined 36-48 h later as described previously (Ho & Babiuk, 1979b).
Inhibition of intercellular spread of virus by antibody Subconfluent monolayers of Hep-2 or Vero cells were infected with CDV at a MOI of 2, incubated at 370 for 4 h prior to trypsinization and removal of the cells from the monolayers. The cells were then washed three times in Hanks's balanced salt solution (HBSS) and incubated for 1 h in MEM + anti-CDV antibody (final titre I/ 50) to neutralize any unadsorbed or uncoated extracellular viruses. The cells were washed three times in MEM + 5% FCS, counted and resuspended to the appropriate cell concentration(s) in MEM prior to cocultivation with Hep-2 cells as described previously (Ho & Babiuk, 1979b). In some experiments a constant amount of CDV antibody (100 TCID5o neutralizing units) was added to cultures containing varying numbers of infected cells while in others a fixed number of infected cells were cultured in different concentrations of antibody. Three days later the monolayers were fixed, stained, and the number of plaques counted. In parallel experiments, canine macrophages were prepared as described previously (Ho & Babiuk, 1 979a). Peripheral blood leucocytes were cultured in vitro. The monolayers were vigorously washed daily for 10 days to remove lymphocytes and nonmacrophage cell types to yield a culture containing >99% macrophages. These cells were then infected with CDV and incubated a further 24 h at 370 to inactivate extracellular virus before the addition of a Hep-2 or Vero cell overlay either in the presence or absence of various concentrations of anti-CDV antibody. The Hep-2 or Vero macrophage co-cultivated cultures were left undisturbed for 3 days before fixing, staining and counting the number of plaques present.
Immune mechanisms against canine distemper II
To determine the kinetics of inhibition of intercellular spread of virus, Hep-2 and Vero cells were infected at a MOI of 2, trypsinized after 4 h of incubation at 370 and cocultivated with Vero or Hep-2 cells. At various time intervals antibody was added to the cultures. Three days later the cultures were terminated and the number of plaques was determined.
Neutralization of cel/fusion factor Vero cells were grown in MEM + 5% FCS in 35 mm tissue culture dishes (Corning No. 25,000) and when the cells became confluent, the culture medium was removed and different volumes of fresh CDV suspension (prepared by serial passages in Hep-2 cells) and dog anti-CDV antiserum or control normal serum were added to each plate. The cells were incubated at 37° in 3% CO2 for 2 h and then trypsinized. Detached cells from triplicate plates were pooled, washed twice in HBSS and diluted with MEM + 5% FCS to 1/5 the original cell concentration. One ml aliquots of each cell suspension were seeded onto 35 mm culture dishes containing a 100 mm2 glass cover slip and the plates were incubated at 370 for about 2 h. When all the cells were attached to the glass surface, the cover slips were removed, the cells were stained with haematoxylineosin stain and the number of cells containing more than one nucleus were counted using a light microscope. Neutralization ofhaemolysin activity Fresh monkey red blood cells were washed 3 x in Alsever's solution and once in phosphate-buffered saline (PBS, 0 1 M Na2HPO4, 0 15 M NaCl, pH 7 2) by centrifugation at 1000 g for 10 min. Packed monkey red blood cells (MRBC) (0 5 ml) were resuspended in 2 ml of PBS and 0 2 ml of [51Cr]-sodium chromate (51Cr, New England Nuclear, Dorval, P.Q.). The cells were incubated at 370 for 90 min with occasional shaking. Following the labelling process, excess 51Cr was removed by repeated washings of the MRBC with PBS. The labelled cells were resuspended in PBS + 01 mm magnesium chloride to a 10% final concentration and 0-2 ml aliquots were dispensed into plastic tissue culture tubes (Falcon No. 2054) containing fresh CDV with or without various concentrations of dog antiserum, control serum or MEM + 5% FCS. The tubes were incubated in a 37° waterbath with constant shaking. After 4 h of incubation, MRBC in each tube were pelleted by centrifugation at I000g for 10 min and half the supernatant was harvested and radioactivity was
767
determined by a gamma spectrophotometer. All tests were performed in triplicate. Antibody-induced modulation of viral surface antigens Vero cells were infected with CDV as described above and when CPE became visible, the cells were removed from the culture dishes by brief exposure to 0.025% trypsin in 0 0I0% EDTA, washed twice in MEM + I0% FCS and resuspended to a concentration of I x 106 cells/ml in plastic tissue culture tubes (Falcon No. 2054). The diluent was foetal calf serum containing 2 mg/ml fluorescein-isothiocyanate (FITC) conjugated dog anti-CDV gammaglobulin. Staining patterns of the cells were observed by fluorescent microscopy after the cells were incubated at different temperatures for various periods of time. Non-infected Vero cells were set up in parallel to serve as negative controls. RESULTS Inhibition of CDV infectivity and virus spread by antibody Since we could easily neutralize CDV with antibody we attempted to determine whether the degree of neutralization was consistent regardless of the cell type used for the neutralization test. Hep-2 cells were chosen because they produced easily visible plaques and Vero cells produced rapid cytopathological changes without plaque formation. Figure 1 illustrates that both systems were useful for demonstrating the neutralizing activity of antibody on extracellular virus.
a
Vb
a-
9 JA
m
