Vol. 13, No. 3 Printed in U.S.A.
INFECTION AND IMMUNITY, Mar. 1976, p. 890-897
Copyright © 1976 American Society for Microbiology
Cross-Reacting Herpes Simplex Virus Antigens in Hamster and Mouse Cells Transformed by Ultraviolet LightInactivated Herpes Simplex Virus Type 2 ANN L. BOYD
Frederick Cancer Research Center, Frederick, Maryland 21701
Received for publication 21 October 1975
Murine and hamster cell lines, each transformed with a different strain of herpes simplex virus (HSV), were examined for cross-reacting antigens by in vitro and in vivo assays. A comparative study by the indirect immunofluorescence technique detected common cross-reacting viral antigens. Cytoplasmic fluorescence patterns were observed in the 333-8-9 hamster line, the H238 murine line, and the H238 clonal lines; these patterns were identical to the fluorescence pattern of HSV-2-infected controls when reacted with HSV antiserum. Tumor rejection studies in the BALB/c host indicated that each cell line provides immunity against a tumorigenic challenge of transformed mouse cells. The H238 clone ECI 3 provided a 53% immunity against itself at an inoculum of 106; the 333-8-9 line supported a 26% immunity. These data demonstrate a common HSV antigenicity between the murine and hamster transformed lines and further indicate that the HSV genome is involved in transformation.
Herpes simplex virus (HSV) is involved in cell transformation, but the transformation characteristics are not as well recognized as in some other virus-transformed systems. Since most animal cells are permissive hosts for HSV production, the transforming potential of the virus has been demonstrated only when the cytopathic effect of the virus was blocked (1, 2, 5-7, 11, 13). All of the resulting transformed lines express HSV antigens (1, 2, 5-7) and/or enzymes (11). One of the first herpes systems reported was the transformation of thymidine kinaseless L (Ltk-) cells after infection with ultraviolet (UV)-inactivated HSV-1 (11). The Ltk- cells acquired HSV-specific thymidine kinase (Ltk+) and expressed several HSV-related antigens. Hamster embryo fibroblasts (HEF) were also transformed after exposure to UV-inactivated HSV-1 or HSV-2. The transformed cell lines derived from the HEF express HSV antigens (6, 7); the sera from hamsters bearing tumors induced by the transformed HEF cells contain neutralizing antibody (6, 7); also, HSV-2-specific messenger ribonucleic acid was detected in the transformed cells by nucleic acid hybridization (4). A BALB/c murine cell line, 238, was transformed after exposure to UV-inactivated HSV-2 (1, 2). The resulting cell line, H238, expresses HSV-related antigens after numerous in vivo and in vitro passages and induces neutralizing
antibody in tumor-bearing hosts (1, 2). Singlecell clones of H238 retain HSV antigenicity, induce tumors at the same dosage as H238 uncloned cells, but do not induce neutralizing antibodies. H238 and single-cell clones of H238 provide protection against subsequent tumor challenge in the homologous BALB/c system (unpublished observations). This study shows that the HSV antigens in the 333-8-9 hamster line and the H238 murine system are common to both systems, as demonstrated by the indirect immunofluorescence (IF) technique and by tumor rejection tests. The expression of HSV antigens in heterologous cell lines transformed by different strains of HSV-2 supports the conclusion that HSV is an oncogenic virus. MATERIALS AND METHODS Cells. The HEF cell lines 333-8-9 and 14-012-8-1, transformed by UV-inactivated HSV-2 (333 strain) and HSV-1 (14-012 strain), respectively, have been described elsewhere (6, 7). The H238 cell line was derived from 238 mouse cells after exposure to UVinactivated HSV-2 (Savage strain). The characteristics of the H238 system have been described (1, 2). Clonal lines from single H238 cells have analogous properties to H238 cells except that neutralizing antibody is not present in tumor-bearing mouse serum (TBS) (unpublished observations). Four cell lines (ECl 3, EA3, A4, and T4) were derived from the H238 line and used in tumor immunity studies. A single cell from the parent, H238, produced a clone 3 890
VOL. 13, 1976
(Cl 3) line, which was used to induce tumors in BALB/c mice; cells from one of the tumors gave rise to the explant clone 3 (ECl 3) line. EA3 and A4 are lines derived from tumors arising after two in vivo passages of a primary H238 tumor. T4 was derived from a tumor induced by the fourth in vivo H238 tumor transplantation. E4 is a simian virus 40 (SV40)-transformed mouse line obtained from C. W. Boone (National Institutes of Health, Bethesda, Md.). MCA-3 is a mouse line derived from an explanted fibrosarcoma induced by methylcholanthrene; the MCA-3 line was also obtained from C. W. Boone. BSC-1 is a continuous green monkey kidney line obtained from the American Type Culture Collection (Rockville, Md.). Rabbit kidney (RK) cultures were obtained from Flow Laboratories (Bethesda, Md.). Serum. A suspension of HSV-1- or HSV-2-infected RK cells (0.1 ml) containing 10f plaque-forming units of HSV per ml was inoculated into two adult New Zealand white rabbits at six distinct sites on the back of each rabbit. Inoculations were repeated weekly for 4 weeks, and an additional intravenous booster of 1 ml of HSV-2-infected RK cells was given at week 6. At week 7, the rabbits were bled by cardiac puncture, and the resultant HSV antiserum was processed and stored at -70 C. Hamster TBS was generously supplied by Fred Rapp (Milton S. Hershey Medical Center, Hershey, Pa.). Three sera were obtained from patients with recurrent genital herpesvirus infections and had HSV2 antibody titers above 1:128 by the complement fixation assay. These sera were kindly provided by Gary Pearson (National Cancer Institute, Bethesda, Md.). Neutralization of HSV. Serum dilutions (0.5 ml) were mixed with 5 x 10" plaque-forming units of HSV per 0.5 ml. The serum-virus mixtures were incubated in a water bath equipped with a rotary shaker for 1 h at 37 C. Subsequently, 0.2-ml samples of the serum-virus mixture were inoculated onto RK cell monolayers in 25-cm2 Falcon flasks in triplicate and adsorbed on a rocker platform for 2 h at room temperature. The cells were then washed with serum-free medium and fed with growth medium. After 4 days of incubation at 37 C in a 10% C02-air atmosphere, the cells were stained with 1% crystal violet in 20% ethanol and washed. The plaques were then counted. IF techniques. The indirect IF technique was used to detect HSV antibodies in TBS. Monolayers of transformed cells were grown on 15-mm2 glass cover slips until confluent, washed three times with warm tris(hydroxymethyl)aminomethane (Tris)buffer, pH 7.4, and then air dried. After fixation in acetone for 10 min, the cells were overlaid with rabbit HSV antiserum or HSV-transformed cell-induced TBS prepared in mouse and hamsters for 30 min at room temperature. The cells were then washed three more times with Tris buffer and exposed to the appropriate gamma globulin conjugated to fluorescein isothiocyanate. The cover slips were washed again three times in warm Tris, air dried, and mounted on slides in 0.1 ml of phosphatebuffered saline-glycerol (1:1). Fluorescence was de-
CROSS-REACTING HSV ANTIGENS
891
tected by using a Leitz microscope equipped with a UV light source. Tumor rejection assay. An in vivo test for cellular immunity, the tumor transplantation rejection assay, originally described by Prehn and Main (12), was modified for the HSV system as follows. HSVtransformed cells (H238 and clones of H238) were X irradiated with a lethal dose of 5,000 rads for 15 min. An inoculum of 106 X-irradiated cells was given subcutaneously into immunocompetent male BALB/ c mice aged 6 to 8 weeks. On day 14, a challenge dose of 10" or 105 live transformed cells was given subcutaneously on the opposite side of the mouse. Tumor incidence was scored by weekly palpation for 60 days. Mice from each group were bled; the tumor size was measured, and tumor sections were examined histologically. TBS was collected and assayed for neutralizing activity by the plaque reduction method. Statistical analysis of the data. The tumor immunity data were statistically analyzed by the Information Systems Group at the Frederick Cancer Research Center (Frederick, Md.), using the Fisher exact test (8). RESULTS
Detection of cross-reacting HSV antigens in hamster and murine transformed cells. The indirect IF technique was used to detect the presence of cross-reacting antigens in the hamster and murine HSV-transformed cell lines (Table 1). Ten percent of the cells from hamster HSV-2-transformed 333-8-9 cells exhibited cytoplasmic fluorescence with rabbit HSV antiserum (Fig. 1). The pattern of cytoplasmic fluorescence in the transformed cells was identical to that observed in RK cells that were infected with HSV-2 harvested 18 h postinfection, and the cells reacted with the same HSV antiseTABLE 1. Comparison of HSV antigen expression in murine and hamster HSV-transformed cells by immunofluorescence Specificity of antiserum Cells"
Rabbit
Rabbit
anti-
anti-
HSV-2
HSV-1
Preimmune
rabbit
serum
Mouse TBS
Hamster TBS
+ + + + 333-8-9 + + 14-012 + + 3+ 4+ 2+ 3+ H238 3+ 2+ 4+ 4+ ECI 3 238 2+ 2+ 2+ 4+ HSV-infected 238 a Cells were grown on 15-mm round glass cover slips, washed, air dried, and fixed for 5 min in cold acetone. The cells were then stained by the indirect immunofluorescence method described in the text. Cells were examined for cytoplasmic fluorescence and scored 4+ if 100% of the cells were positive; 3+ if =75% were positive; 2+ if 50% were positive; 1+ if 10 to 25% were positive; and ± if
INFECTION AND IMMUNITY, Mar. 1976, p. 890-897
Copyright © 1976 American Society for Microbiology
Cross-Reacting Herpes Simplex Virus Antigens in Hamster and Mouse Cells Transformed by Ultraviolet LightInactivated Herpes Simplex Virus Type 2 ANN L. BOYD
Frederick Cancer Research Center, Frederick, Maryland 21701
Received for publication 21 October 1975
Murine and hamster cell lines, each transformed with a different strain of herpes simplex virus (HSV), were examined for cross-reacting antigens by in vitro and in vivo assays. A comparative study by the indirect immunofluorescence technique detected common cross-reacting viral antigens. Cytoplasmic fluorescence patterns were observed in the 333-8-9 hamster line, the H238 murine line, and the H238 clonal lines; these patterns were identical to the fluorescence pattern of HSV-2-infected controls when reacted with HSV antiserum. Tumor rejection studies in the BALB/c host indicated that each cell line provides immunity against a tumorigenic challenge of transformed mouse cells. The H238 clone ECI 3 provided a 53% immunity against itself at an inoculum of 106; the 333-8-9 line supported a 26% immunity. These data demonstrate a common HSV antigenicity between the murine and hamster transformed lines and further indicate that the HSV genome is involved in transformation.
Herpes simplex virus (HSV) is involved in cell transformation, but the transformation characteristics are not as well recognized as in some other virus-transformed systems. Since most animal cells are permissive hosts for HSV production, the transforming potential of the virus has been demonstrated only when the cytopathic effect of the virus was blocked (1, 2, 5-7, 11, 13). All of the resulting transformed lines express HSV antigens (1, 2, 5-7) and/or enzymes (11). One of the first herpes systems reported was the transformation of thymidine kinaseless L (Ltk-) cells after infection with ultraviolet (UV)-inactivated HSV-1 (11). The Ltk- cells acquired HSV-specific thymidine kinase (Ltk+) and expressed several HSV-related antigens. Hamster embryo fibroblasts (HEF) were also transformed after exposure to UV-inactivated HSV-1 or HSV-2. The transformed cell lines derived from the HEF express HSV antigens (6, 7); the sera from hamsters bearing tumors induced by the transformed HEF cells contain neutralizing antibody (6, 7); also, HSV-2-specific messenger ribonucleic acid was detected in the transformed cells by nucleic acid hybridization (4). A BALB/c murine cell line, 238, was transformed after exposure to UV-inactivated HSV-2 (1, 2). The resulting cell line, H238, expresses HSV-related antigens after numerous in vivo and in vitro passages and induces neutralizing
antibody in tumor-bearing hosts (1, 2). Singlecell clones of H238 retain HSV antigenicity, induce tumors at the same dosage as H238 uncloned cells, but do not induce neutralizing antibodies. H238 and single-cell clones of H238 provide protection against subsequent tumor challenge in the homologous BALB/c system (unpublished observations). This study shows that the HSV antigens in the 333-8-9 hamster line and the H238 murine system are common to both systems, as demonstrated by the indirect immunofluorescence (IF) technique and by tumor rejection tests. The expression of HSV antigens in heterologous cell lines transformed by different strains of HSV-2 supports the conclusion that HSV is an oncogenic virus. MATERIALS AND METHODS Cells. The HEF cell lines 333-8-9 and 14-012-8-1, transformed by UV-inactivated HSV-2 (333 strain) and HSV-1 (14-012 strain), respectively, have been described elsewhere (6, 7). The H238 cell line was derived from 238 mouse cells after exposure to UVinactivated HSV-2 (Savage strain). The characteristics of the H238 system have been described (1, 2). Clonal lines from single H238 cells have analogous properties to H238 cells except that neutralizing antibody is not present in tumor-bearing mouse serum (TBS) (unpublished observations). Four cell lines (ECl 3, EA3, A4, and T4) were derived from the H238 line and used in tumor immunity studies. A single cell from the parent, H238, produced a clone 3 890
VOL. 13, 1976
(Cl 3) line, which was used to induce tumors in BALB/c mice; cells from one of the tumors gave rise to the explant clone 3 (ECl 3) line. EA3 and A4 are lines derived from tumors arising after two in vivo passages of a primary H238 tumor. T4 was derived from a tumor induced by the fourth in vivo H238 tumor transplantation. E4 is a simian virus 40 (SV40)-transformed mouse line obtained from C. W. Boone (National Institutes of Health, Bethesda, Md.). MCA-3 is a mouse line derived from an explanted fibrosarcoma induced by methylcholanthrene; the MCA-3 line was also obtained from C. W. Boone. BSC-1 is a continuous green monkey kidney line obtained from the American Type Culture Collection (Rockville, Md.). Rabbit kidney (RK) cultures were obtained from Flow Laboratories (Bethesda, Md.). Serum. A suspension of HSV-1- or HSV-2-infected RK cells (0.1 ml) containing 10f plaque-forming units of HSV per ml was inoculated into two adult New Zealand white rabbits at six distinct sites on the back of each rabbit. Inoculations were repeated weekly for 4 weeks, and an additional intravenous booster of 1 ml of HSV-2-infected RK cells was given at week 6. At week 7, the rabbits were bled by cardiac puncture, and the resultant HSV antiserum was processed and stored at -70 C. Hamster TBS was generously supplied by Fred Rapp (Milton S. Hershey Medical Center, Hershey, Pa.). Three sera were obtained from patients with recurrent genital herpesvirus infections and had HSV2 antibody titers above 1:128 by the complement fixation assay. These sera were kindly provided by Gary Pearson (National Cancer Institute, Bethesda, Md.). Neutralization of HSV. Serum dilutions (0.5 ml) were mixed with 5 x 10" plaque-forming units of HSV per 0.5 ml. The serum-virus mixtures were incubated in a water bath equipped with a rotary shaker for 1 h at 37 C. Subsequently, 0.2-ml samples of the serum-virus mixture were inoculated onto RK cell monolayers in 25-cm2 Falcon flasks in triplicate and adsorbed on a rocker platform for 2 h at room temperature. The cells were then washed with serum-free medium and fed with growth medium. After 4 days of incubation at 37 C in a 10% C02-air atmosphere, the cells were stained with 1% crystal violet in 20% ethanol and washed. The plaques were then counted. IF techniques. The indirect IF technique was used to detect HSV antibodies in TBS. Monolayers of transformed cells were grown on 15-mm2 glass cover slips until confluent, washed three times with warm tris(hydroxymethyl)aminomethane (Tris)buffer, pH 7.4, and then air dried. After fixation in acetone for 10 min, the cells were overlaid with rabbit HSV antiserum or HSV-transformed cell-induced TBS prepared in mouse and hamsters for 30 min at room temperature. The cells were then washed three more times with Tris buffer and exposed to the appropriate gamma globulin conjugated to fluorescein isothiocyanate. The cover slips were washed again three times in warm Tris, air dried, and mounted on slides in 0.1 ml of phosphatebuffered saline-glycerol (1:1). Fluorescence was de-
CROSS-REACTING HSV ANTIGENS
891
tected by using a Leitz microscope equipped with a UV light source. Tumor rejection assay. An in vivo test for cellular immunity, the tumor transplantation rejection assay, originally described by Prehn and Main (12), was modified for the HSV system as follows. HSVtransformed cells (H238 and clones of H238) were X irradiated with a lethal dose of 5,000 rads for 15 min. An inoculum of 106 X-irradiated cells was given subcutaneously into immunocompetent male BALB/ c mice aged 6 to 8 weeks. On day 14, a challenge dose of 10" or 105 live transformed cells was given subcutaneously on the opposite side of the mouse. Tumor incidence was scored by weekly palpation for 60 days. Mice from each group were bled; the tumor size was measured, and tumor sections were examined histologically. TBS was collected and assayed for neutralizing activity by the plaque reduction method. Statistical analysis of the data. The tumor immunity data were statistically analyzed by the Information Systems Group at the Frederick Cancer Research Center (Frederick, Md.), using the Fisher exact test (8). RESULTS
Detection of cross-reacting HSV antigens in hamster and murine transformed cells. The indirect IF technique was used to detect the presence of cross-reacting antigens in the hamster and murine HSV-transformed cell lines (Table 1). Ten percent of the cells from hamster HSV-2-transformed 333-8-9 cells exhibited cytoplasmic fluorescence with rabbit HSV antiserum (Fig. 1). The pattern of cytoplasmic fluorescence in the transformed cells was identical to that observed in RK cells that were infected with HSV-2 harvested 18 h postinfection, and the cells reacted with the same HSV antiseTABLE 1. Comparison of HSV antigen expression in murine and hamster HSV-transformed cells by immunofluorescence Specificity of antiserum Cells"
Rabbit
Rabbit
anti-
anti-
HSV-2
HSV-1
Preimmune
rabbit
serum
Mouse TBS
Hamster TBS
+ + + + 333-8-9 + + 14-012 + + 3+ 4+ 2+ 3+ H238 3+ 2+ 4+ 4+ ECI 3 238 2+ 2+ 2+ 4+ HSV-infected 238 a Cells were grown on 15-mm round glass cover slips, washed, air dried, and fixed for 5 min in cold acetone. The cells were then stained by the indirect immunofluorescence method described in the text. Cells were examined for cytoplasmic fluorescence and scored 4+ if 100% of the cells were positive; 3+ if =75% were positive; 2+ if 50% were positive; 1+ if 10 to 25% were positive; and ± if
