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Isolation of High and Low Molecular Weight Components from Chicken Sera That Have Rous Sarcoma Virus Neutralizing Activity1
BREEDING AND GENETICS Isolation of High and Low Molecular Weight Components from Chicken Sera That Have Rous Sarcoma Virus Neutralizing Activity1 C. E. WHITFILL,2 J. ALLEN,3 N. R. GYLES,2 J. THOMA3 and L. T. PATTERSON2 Department of Animal Sciences and Department of Chemistry, University of Arkansas, Fayetteville, AR 72701 and Antaeus Institute, Fayetteville, AR 72701 (Received for publication February 17, 1981)
INTRODUCTION Gyles et al. (1968) and Gyles and Brown (1971) found that regression of Rous sarcoma virus (RSV)-induced tumors is under genetic control in chickens. Further, Schierman et al. (1977) and Collins et al. (1977) have identified a single dominantly inherited gene, designated R-Rs-.l, located outside the B blood locus, which appears to be the primary genetic factor regulating regression in chickens. Differences between the regressor and progressor chickens in early recognition and speed and intensity of response by their immunological systems may be an important key to regression. Gyles et al. (1967) found that the time required for a tumor to be initiated from wing-web inoculation of RSV varied among genetic lines and among individuals within a genetic line. Gyles et al. (1977) discovered that the immune response to a secondary RSV challenge with either RSV or Rous sarcoma
1 Published with the approval of the Director of the Arkansas Agricultural Experiment Station. 2 Department of Animal Sciences. 3 Department of Chemistry and Antaeus Institute.
tumor homogenate (RSTH) in the opposite wing-web was met by an earlier immune response in the R-line than in the Pr-line, but that both lines could mount a response to the second challenge. In this paper the time dependence of appearance and disappearance of RSV neutralizing activity of HMW-I and LMW-II factors present in Arkansas regressor and progressor lines is reported. MATERIALS AND METHODS Experimental Animals. The regressor chickens used for these experiments were from two lines genetically selected for their ability to regress Rous-sarcomas by Gyles et al. (1967) at the Arkansas Experimental Station and designated the R-line. The progressor chickens used for these experiments were from the White Leghorn line maintained at the Arkansas Experimental Station and designated the Pr-line. Estimated inbreeding in R-line is 39 and 43% in Pr-line. The frequency of the R-Rs-1 gene in the R-line is estimated to be .91. Standard Inoculum of Rous sarcoma Virus (RSV). The RSV used was prepared for the National Cancer Institute, National Institute of Health by University Laboratories, Inc., High-
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ABSTRACT Blood sera components from Arkansas regressor line (R-line) and progressor line (Pr-line) chickens are compared for the first time for Rous sarcoma virus neutralizing activity. Sera was fractionated by Sephadex G-100 gel filtration into a high molecular weight fraction I (HMW-I) and a low molecular weight fraction II (LMW-II) component (HMW-I > 14,000 daltons, LMW-II < 5,000 daltons). Both fractions from each line of chickens exhibit activity against Rous sarcoma virus (RSV) judged by a wing web assay. Both HMW-I (principally antibodies) and LMW-II neutralized RSV when obtained from hyperimmune R-chickens and Pr-chickens with large progressing tumors. However, HMW-I and LMW-II obtained from R- or Pr-chickens before challenge contain no RSV neutralizing activity. The novel low molecular weight fraction II disappeared from the sera of R-line chickens 2 weeks after tumor regression, whereas the HMW-I persisted after tumor regression. (Key words: Rous sarcoma virus, chicken, sera, neutralize) 1982 Poultry Science 61:1573-1578
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frozen to —50 C, freeze-dried, and stored at —25 C. Before experimentation, LMW-II was redissolved with distilled water resulting in a concentration of 5 mg protein/ml in 1.0 M NaCl, .2 M citrate, pH 8.2. Protein concentrations were determined by method of Lowry etal. (1951). Effect of Pretreatment of RSV with HMW-I and LMW-II. Each group of Pr- chickens received an injection consisting of either a standard inoculum of RSV preincubated with 120 mg of HMW-I in 1 ml buffer (.15 M NaCl, .01 M citrate, pH 7.5) or a standard inoculum of RSV preincubated with 5 mg of LMW-II in .5 ml buffer (1.0 M NaCl, .2 M citrate, pH 8.2). Each control group of Pr- chickens received an injection of either a standard inoculum of RSV plus 1 ml buffer (.15 M NaCl, .01 M citrate, pH 7.5) or a standard inoculum of RSV plus .5 ml of buffer (1.0 M NaCl, .2 M citrate, pH 8.2). Treatment and control inoculums were preincubated with occasional stirring at 5 C for 3 hr before subcutaneous injection into Pr-line chickens and subsequent monitoring of tumor growth. RESULTS
Fractionation of Chicken Sera into HMW-I and LMW-II Components. A typical elution profile for chicken sera from a Sephadex G-100 column is shown in Figure 1. The first fraction, containing the high molecular weight sera components, has molecular weights greater than 14,000 and the second fraction, containing the low molecular weight sera components, has molecular weights of 14,000 or less based on their elution volume relative to that of lysozyme. Dependence of HMW-I and LMW-II on RSV Challenge. To test whether R- and Pr-line chickens were both capable of manufacturing HMW-I and LMW-II activity in response to RSV challenge, the anti-RSV activity was tested from untreated and sensitized chickens. Sera samples were obtained from normal R- and Pr-chickens before RSV challenge and fractionated into HMW-I and LMW-II and each fraction tested for anti-RSV activity. To test for the presence of HMW-I and LMW-II components at a certain time after RSV challenge, samples of sera were withdrawn 32 days (approximately 2 weeks after complete tumor remission) after RSV inoculation in R-chickens and 20 days after RSV inoculation in Prchickens and fractionated into HMW-I and
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land Park, NJ. Bryan High Titer Strain RSV was used and identified as lot number TV-26 with an infectivity titer of 1 0 7 ' 4 plaque-forming units/ml. The RSV was stored at —50 C in sealed ampules and before use was thawed rapidly in a 37 C water bath and diluted 10 5 in 5 C Hanks' balanced salt solution. Inoculation of birds was accomplished by injecting a standard inoculum of .2 ml of diluted RSV subcutaneously into the left wing-web. Preparation of R-Line Hyperimmune Sera. R-Hne chickens were challenged with a standard inoculum of RSV, and after complete tumor regression as measured by visible disappearance of tumors, birds were injected once weekly for at least 3 weeks with a standard inoculum of RSTH booster. Five days after the last booster RSTH injection, 20 ml of blood was removed from each chicken by cardiac puncture and allowed to clot for 1 hr at room temperature. Clotted blood was sedimented at 3000 X j at room temperature in a Sorvall centrifuge for 10 min and hyperimmune sera was removed and stored at 5 C. Fractionation of Chicken Sera by Sephadex G-100 Column Chromatography. To examine for the presence of HMW I and LMW II in the two genetic lines of chickens, samples of blood were withdrawn at various times after challenge with virus. Sera samples were fractionated into the two components, and each was tested for virus neutralizing activity. Whole sera samples from the R- and Pr-lines of chickens were separately applied in a volume of 125 ml to a 6 X 55 cm Sephadex G-100 column and run at room temperature with buffer (.01 M citrate, .05 M NaCl, pH 7.2) at a flow rate of 300 ml/hr. Molecular weight standards, blue dextran (2,000,000 daltons, bovine hemoglobin (64,500 daltons), lysozyme (14,000 daltons), and potassium ferricyanide (329.26 daltons) were used to calibrate the Sephadex G-100 column. Fractionation of Sera Components into High Molecular Weight Fraction I (HMW-I) and Low Molecular Weight Fraction II (LMW-II). When 125 ml of sera were loaded onto a 6 X 55 cm Sephadex G-100 column, the HMW-I eluted in a volume of 500 ml. This fraction was concentrated at 5 C by dialysis in 15% polyethylene glycol in buffer (.15 M NaCl, .01 M citrate, pH 7.5) to a final concentration of 120 mg/ml and was stored at 5 C after removal of polyethylene glycol by dialysis against buffer. The LMW-II serum components, which eluted from the column in a volume of 700 ml, were
CHICKEN SERA TO NEUTRALIZE RSV
The HMW-I isolated from either R-line or Pr-line chicken sera before RSV challenge has no anti-RSV activity (Fig. 2A) because it did not prevent tumor initiation, and the tumors grew progressively until essentially all the chickens had died. The differences among controls that died (80%) and those treated with R-line HMW-I that died (80%) and those treated with Pr-line HMW-I (100%) are not significant as determined by Student's t test (Davies, 1954). It can be concluded that anti-RSV
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DAYS POST INITIAL RSV INOCULATION FIG. 2. Preincubation of RSV with HMW-I (A) and LMW-II (B) followed by injection into Pr-line chicken wing-webs. HMW-I and LMW-II were isolated from R-line and Pr-line sera (obtained before RSV 8 9 10 II 12 13 14 15 16 17 challenge) by Sephadex G-100 column chromatography. In A, ( • ) represents treated group ELUTION VOLUME (ml) where each chicken received an injection containing x 100 RSV plus 120 mg HMW-I from R-line unsensitized chicken sera, (- - A - -) represents treated group where FIG. 1. Sephadex G-100 column chromatography each chicken received an injection containing RSV elution profile of R-line hyperimmune sera fractions plus 120 mg HMW-I from Pr-line unsensitized chicken and molecular weight standards. R-line hyperimmune sera in a sample volume of 125 ml was applied to sera and ( o ) represents control group where the column and the sera components with molecular each chicken received RSV only. In B, (- - -• ) weights greater than 14,000 that eluted between represents treated group where each Pr-chicken 200 and 700 ml were collectively pooled and~ conreceived an injection containing RSV plus 5 mg centrated (HMW-I); sera components with molecular LMW-II from R-line unsensitized chicken sera, weights of 14,000 or less that eluted between 800 ( - - * - - ) represents treated group where each chicken and 1500 ml were collectively pooled and concenreceived an injection containing RSV plus 5 mg trated (LMW-II). All other sera samples were treated LMW-II from Pr-line unsensitized chicken sera, and ( o ) represents control group where each similarly. Symbols: ( o ), elution profile chicken received RSV only. Numbers in bold face of R-line hyperimmune sera fractionation; ( * ), print represent the number of chickens remaining rechromatography of an a l i q u o t of LMW-II; alive at various times post initial RSV inoculation ( • ), profile of molecular weight standards in treated and control groups. Tumor scores decline (A, blue dextran; B, bovine hemoglobin; C, lysozyme; after sensitive chickens die. D, potassium ferricyanide).
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LMW-II components. Each fraction was tested for anti-RSV activity by incubation with RSV and subsequent injection into the wing-web of 8-week-old Pr-chickens. Tumors were scored (Gyles et al, 1977) periodically and the results of these experiments are recorded in Figures 2, 3, and 4. Average tumor score after 30 days declined because the susceptible Pr-chickens had died and the more resistant chickens had begun to regress their tumors. There is no evidence for virus neutralizing activity in either HMW I or LMW II from unsensitized chickens. The sizes of the group of chickens at the start and at various times during the experiment are given in boldface letters in the figures. In these experiments, HMW-I and LMW-II clearly protect Pr-chicken when the samples are isolated from sensitized animals.
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antibodies are not present in the HMW-I components of the sera in either the R- or Prflocks of unchallenged chickens.
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Figure 3A shows the effect of R-line HMW-I prepared from chicken sera 32 days postinoculation (after regression) and the effect of Pr-line HMW-I prepared from sera 20 days postinoculation. R-line HMW-I completely suppressed tumorgenesis of the RSV after
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FIG. 3. Preincubation of RSV with HMW-I (A) and LMW-II (B) followed by injection into Pr-line chicken wing-webs. HMW-I and LMW-II were isolated from R-line and Pr-line immune sera by Sephadex G-100 column chromatography. In A, (- - -•- - -) represents treated group where each chicken received an injection containing RSV plus 120 mg HMW-I from R-line immune sera obtained 2 weeks after tumor regression (32 days post RSV challenge), ( - - * - - ) represents treated group where each chicken received an injection containing RSV plus 120 mg HMW-I from Pr-line immune sera obtained while chickens were carrying #4 size tumors (20 days post RSV challenge) and ( o ) represents control group where each chicken received an injection containing RSV only. In B, ( • ) represents treated group where each chicken received an injection containing RSV plus 5 mg LMW-II from R-line immune sera obtained 32 days post RSV challenge, ( - - * - - ) represents treated group where each chicken received an injection containing RSV plus 5 mg LMW-II from Pr-line immune sera obtained 20 days post RSV challenge, and ( o ) represents control group where each chicken received RSV only. Numbers in boldface print as in Figure 2.
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FIG. 4. Preincubation of RSV with HMW-I and LMW-II followed by injection in Pr-line chicken wing webs. HWM-I and LMW-II were isolated from R-line hyperimmune sera by Sephadex G-100 column chromatography. In the figure, ( • ) represents treated group where each chicken received an injection containing RSV plus 120 mg HMW-I, ( - - * - - ) represents treated group where each chicken received an injection containing RSV plus 5 mg LMW-II and ( o ) represents control group where each chicken received RSV only. Numbers in boldface print as in Figure 2.
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Similarly, neither the R- nor Pr-line LMW-II from sera from unchallenged chickens exhibited any anti-RSV activity against a standard inoculum of RSV. The pertinent data are recorded in Figure 2B. In the group treated with RSV preincubated with R-line LMW-II, tumors were initiated and grew progressively to # 4 scores before regressing or showing definite signs of regression in 43% of 33 chickens. The remaining 57% treated died or had #4 scores 75 days postinjection. Thirty percent of the control chickens regressed tumors, which was not significantly different from the treated group. Figure 2B also shows that preincubation with RSV of Pr-line LMW-II from sera of unchallenged chickens does not inactivate the RSV. Seventy-eight percent of the treated chickens injected died and 22% regressed, and there was no significant difference between treated and control groups as judged by Student's t test (Davies, 1954).
CHICKEN SERA TO NEUTRALIZE RSV
As expected (see Fig. 4), the HMW-I from hyperimmune sera from R-line chickens was capable of completely arresting the activity of RSV. None of the injected chickens developed tumors, whereas 80% of the control group were dead or carrying # 4 tumors 32 days postinoculation (P<.01). Reimmunization of the R-chickens with RSTH (hyperimmune chickens) restimulates the production of anti RSV-LMW-II as shown in Figure 4. In the treated group, 35% of the chickens did not develop tumors, whereas die other 55% regressed or were regressing tumors during the 32 days observation period. At the end of the experiment 90% of the treated group had no tumors whereas 80% of the control group had died. The difference is significant
(P<.01). This is a very interesting result, because LMW-II components are not present in R-line chicken immune sera 32 days post-RSV (after tumor regression) but can be restimulated by a booster inoculation of RSV or tumor homogenate.
DISCUSSION
The most interesting aspect of this study was the observation of a low molecular weight component in both progressor and regressor sera that possessed viral neutralizing activity. The origin of the anti-RSV LMW-II component is unknown, whereas HMW-I is largely or exclusively immunoglobulin (Allen, 1978). It cannot be detected by immunoelectrophoresis with either anti-chicken IgM or IgG antisera. The LMW-II component disappeared 2 weeks after tumor regression in the R-chickens (32 days post-RSV challenge), reappeared upon subsequent rechallenge in the R-line chickens, and just began to appear in high levels in the Pr-chickens at 20 days after RSV challenge when the chickens were carrying large tumors. It is intriguing that cytotoxic T-cells apparently disappear during the regressive phase of tumor development, which makes them attractive candidates as LMW-II producers. The LMW-II and cytotoxic T cells' disappearance after regression can be restimulated by tumor homogenate and immunoglobulins titers remain high for several months (Fink and Rauscher, 1961). These observations also suggest that the LMW-II is not an immunoglobulin fragment. The LMW-II does not stain on SDS-polyacrylamide electrophoresis (data not shown). The partition coefficient of die LMW-II substance is unity on the Sephadex G-100 column so that we estimate its molecular weight is less than 5,000 daltons (Pharmacia Fine Chemicals, 1975). The mechanism of action of LMW-II on virus is not known, but it could be inactivating RSV from transformed cells (Wainberg et al., 1979). The possibility, of course, exists that the LMW-II has general antiviral activity and acts by inhibiting nucleic acid replication (Frume et al, 1979; Burzynski^a/., 1976). ACKNOWLEDGMENTS We thank Van Thompson, Omer McConnell, Olen Dunaway, and Paul Teague for their friendly and constant help at the University of
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preincubation. By 41 days postinoculation, none of the treated chickens had developed tumors, whereas 87% of the control animals had died (P<.01) from Rous sarcomas. The Pr-line HMW-I also had anti-RSV activity. Thirty percent of the animals challenged with RSV preincubated with Pr-line HMW-I developed small tumors, but all had completely regressed within 41 days after challenge and significantly differ from the controls (P<.01). The difference in tumor takes between the R-line HMW-I (0%) and the Pr-line HMW-I (30%) treated RSV is not significant but hints at a greater RSV neutralizing activity in the R-sera. The levels of anti-RSV activity in the LMWII from R-chickens taken at 32 days postinoculation and from Pr-chickens taken 20 days post-RSV challenge were unexpected (Fig. 3B). The R-line LMW-II activity had disappeared, because 80% of the treated chickens died (received RSV plus R-line LMW-II) with progressive tumors and only 20% showed signs of tumor regression. The treated group did not differ significantly from the control group where 67% had died and the rest were carrying #4 tumors at the termination of the experiment 79 days postchallenge. However, pretreatment of RSV with LMW-II from Pr-birds carrying heavy tumor burdens (score 4) was effective in reducing the infectivity of the virus. In the treated group, 56% of the chickens did not develop visible tumors and the remaining 44% either regressed tumors or were showing definite signs of tumor regression at the termination of the experiment. By day 79 postchallenge, 88% of the birds had no apparent tumor and differed from the control (P<.01).
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Arkansas poultry farrp. Without their help, this work would not have been possible. REFERENCES
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Allen, J., 1978. Studies on the Effect of Purified Rous Sarcoma Virus Specific Antibody on Rous Sarcoma Regression in Chickens. Univ. Arkansas, Fayetteville, AR. Burzynski, S. R., 1976. Antineoplastins: Biochemical defense against cancer. Physiol. Chem. Phys. 8:275-279. Collins, W. M., W. E. Briles, R. M. Zsigray, W. R. Dunlop, A. Corbett, K. K. Clark, J. R. Marks, and T. P. McGrail, 1977. The B locus (MHC) in the chicken: Association with the fate of RSVinduced tumors. Immunogenetics 5:333 — 343. Davies, O. L., 1954. Statistical Methods in Research and Production. Oliver and Boyd, London. Fink, M., and F. Rauscher, 1961. A simple method for potent chicken anti-Rous sarcoma virus serum. J. Natl. Cancer Inst. 26:519-522. Frume, L., A. Mattioli, and C. Busi, 1979. An endog e n o u s inhibitor of fibroblast proliferation hinders Simian virus 40 replication. Naturwissenschaften 66:265—266. Gyles, N. R., and C. J. Brown, 1971. Selection in chickens for retrogression of tumors caused by Rous sarcoma virus. Poultry Sci. 50:901—905.
Gyles, N. R., M. Blythe, P. Test, A. Bowanki, and C. J. Brown. 1977. Immune response in progressor and regressor strains of chickens at specific intervals after primary challenge with Rous sarcoma virus. Poultry Sci. 56:758-766. Gyles, N. R., J. L. Miley, and C. J. Brown, 1967. The response of resistance and susceptible strains of chickens and their F, and F 2 crosses to subcutaneous inoculations with Rous sarcoma virus. Poultry Sci. 46:465-472. Gyles, N. R., B. R. Stewart, and C. J. Brown, 1968. Mechanisms of genetic resistance in trie chicken to Rous sarcoma virus. Poultry Sci. 47:430-450. Lowry, D. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 1 9 3 : 2 6 5 275. Pharmacia Fine Chemicals. 1975. Sephadex gel filtration in theory and practice. Upplands Grafiska AB, Uppsala, Sweden. Schierman, L. W., D. H. Watanabe, and R. A. McBride, 1977. Genetic control of Rous sarcoma regression in chickens: Linkage with the major histocompatibility complex. Immunogenetics 5: .325-332. Wainberg, M. A., B. Buss, R. Wahi, and E. Israel, 1979. The thymic dependence of cell-mediated immunity to avian sarcomas in chickens. Cell Immunol. 45:344-355.
INTRODUCTION Gyles et al. (1968) and Gyles and Brown (1971) found that regression of Rous sarcoma virus (RSV)-induced tumors is under genetic control in chickens. Further, Schierman et al. (1977) and Collins et al. (1977) have identified a single dominantly inherited gene, designated R-Rs-.l, located outside the B blood locus, which appears to be the primary genetic factor regulating regression in chickens. Differences between the regressor and progressor chickens in early recognition and speed and intensity of response by their immunological systems may be an important key to regression. Gyles et al. (1967) found that the time required for a tumor to be initiated from wing-web inoculation of RSV varied among genetic lines and among individuals within a genetic line. Gyles et al. (1977) discovered that the immune response to a secondary RSV challenge with either RSV or Rous sarcoma
1 Published with the approval of the Director of the Arkansas Agricultural Experiment Station. 2 Department of Animal Sciences. 3 Department of Chemistry and Antaeus Institute.
tumor homogenate (RSTH) in the opposite wing-web was met by an earlier immune response in the R-line than in the Pr-line, but that both lines could mount a response to the second challenge. In this paper the time dependence of appearance and disappearance of RSV neutralizing activity of HMW-I and LMW-II factors present in Arkansas regressor and progressor lines is reported. MATERIALS AND METHODS Experimental Animals. The regressor chickens used for these experiments were from two lines genetically selected for their ability to regress Rous-sarcomas by Gyles et al. (1967) at the Arkansas Experimental Station and designated the R-line. The progressor chickens used for these experiments were from the White Leghorn line maintained at the Arkansas Experimental Station and designated the Pr-line. Estimated inbreeding in R-line is 39 and 43% in Pr-line. The frequency of the R-Rs-1 gene in the R-line is estimated to be .91. Standard Inoculum of Rous sarcoma Virus (RSV). The RSV used was prepared for the National Cancer Institute, National Institute of Health by University Laboratories, Inc., High-
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ABSTRACT Blood sera components from Arkansas regressor line (R-line) and progressor line (Pr-line) chickens are compared for the first time for Rous sarcoma virus neutralizing activity. Sera was fractionated by Sephadex G-100 gel filtration into a high molecular weight fraction I (HMW-I) and a low molecular weight fraction II (LMW-II) component (HMW-I > 14,000 daltons, LMW-II < 5,000 daltons). Both fractions from each line of chickens exhibit activity against Rous sarcoma virus (RSV) judged by a wing web assay. Both HMW-I (principally antibodies) and LMW-II neutralized RSV when obtained from hyperimmune R-chickens and Pr-chickens with large progressing tumors. However, HMW-I and LMW-II obtained from R- or Pr-chickens before challenge contain no RSV neutralizing activity. The novel low molecular weight fraction II disappeared from the sera of R-line chickens 2 weeks after tumor regression, whereas the HMW-I persisted after tumor regression. (Key words: Rous sarcoma virus, chicken, sera, neutralize) 1982 Poultry Science 61:1573-1578
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frozen to —50 C, freeze-dried, and stored at —25 C. Before experimentation, LMW-II was redissolved with distilled water resulting in a concentration of 5 mg protein/ml in 1.0 M NaCl, .2 M citrate, pH 8.2. Protein concentrations were determined by method of Lowry etal. (1951). Effect of Pretreatment of RSV with HMW-I and LMW-II. Each group of Pr- chickens received an injection consisting of either a standard inoculum of RSV preincubated with 120 mg of HMW-I in 1 ml buffer (.15 M NaCl, .01 M citrate, pH 7.5) or a standard inoculum of RSV preincubated with 5 mg of LMW-II in .5 ml buffer (1.0 M NaCl, .2 M citrate, pH 8.2). Each control group of Pr- chickens received an injection of either a standard inoculum of RSV plus 1 ml buffer (.15 M NaCl, .01 M citrate, pH 7.5) or a standard inoculum of RSV plus .5 ml of buffer (1.0 M NaCl, .2 M citrate, pH 8.2). Treatment and control inoculums were preincubated with occasional stirring at 5 C for 3 hr before subcutaneous injection into Pr-line chickens and subsequent monitoring of tumor growth. RESULTS
Fractionation of Chicken Sera into HMW-I and LMW-II Components. A typical elution profile for chicken sera from a Sephadex G-100 column is shown in Figure 1. The first fraction, containing the high molecular weight sera components, has molecular weights greater than 14,000 and the second fraction, containing the low molecular weight sera components, has molecular weights of 14,000 or less based on their elution volume relative to that of lysozyme. Dependence of HMW-I and LMW-II on RSV Challenge. To test whether R- and Pr-line chickens were both capable of manufacturing HMW-I and LMW-II activity in response to RSV challenge, the anti-RSV activity was tested from untreated and sensitized chickens. Sera samples were obtained from normal R- and Pr-chickens before RSV challenge and fractionated into HMW-I and LMW-II and each fraction tested for anti-RSV activity. To test for the presence of HMW-I and LMW-II components at a certain time after RSV challenge, samples of sera were withdrawn 32 days (approximately 2 weeks after complete tumor remission) after RSV inoculation in R-chickens and 20 days after RSV inoculation in Prchickens and fractionated into HMW-I and
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land Park, NJ. Bryan High Titer Strain RSV was used and identified as lot number TV-26 with an infectivity titer of 1 0 7 ' 4 plaque-forming units/ml. The RSV was stored at —50 C in sealed ampules and before use was thawed rapidly in a 37 C water bath and diluted 10 5 in 5 C Hanks' balanced salt solution. Inoculation of birds was accomplished by injecting a standard inoculum of .2 ml of diluted RSV subcutaneously into the left wing-web. Preparation of R-Line Hyperimmune Sera. R-Hne chickens were challenged with a standard inoculum of RSV, and after complete tumor regression as measured by visible disappearance of tumors, birds were injected once weekly for at least 3 weeks with a standard inoculum of RSTH booster. Five days after the last booster RSTH injection, 20 ml of blood was removed from each chicken by cardiac puncture and allowed to clot for 1 hr at room temperature. Clotted blood was sedimented at 3000 X j at room temperature in a Sorvall centrifuge for 10 min and hyperimmune sera was removed and stored at 5 C. Fractionation of Chicken Sera by Sephadex G-100 Column Chromatography. To examine for the presence of HMW I and LMW II in the two genetic lines of chickens, samples of blood were withdrawn at various times after challenge with virus. Sera samples were fractionated into the two components, and each was tested for virus neutralizing activity. Whole sera samples from the R- and Pr-lines of chickens were separately applied in a volume of 125 ml to a 6 X 55 cm Sephadex G-100 column and run at room temperature with buffer (.01 M citrate, .05 M NaCl, pH 7.2) at a flow rate of 300 ml/hr. Molecular weight standards, blue dextran (2,000,000 daltons, bovine hemoglobin (64,500 daltons), lysozyme (14,000 daltons), and potassium ferricyanide (329.26 daltons) were used to calibrate the Sephadex G-100 column. Fractionation of Sera Components into High Molecular Weight Fraction I (HMW-I) and Low Molecular Weight Fraction II (LMW-II). When 125 ml of sera were loaded onto a 6 X 55 cm Sephadex G-100 column, the HMW-I eluted in a volume of 500 ml. This fraction was concentrated at 5 C by dialysis in 15% polyethylene glycol in buffer (.15 M NaCl, .01 M citrate, pH 7.5) to a final concentration of 120 mg/ml and was stored at 5 C after removal of polyethylene glycol by dialysis against buffer. The LMW-II serum components, which eluted from the column in a volume of 700 ml, were
CHICKEN SERA TO NEUTRALIZE RSV
The HMW-I isolated from either R-line or Pr-line chicken sera before RSV challenge has no anti-RSV activity (Fig. 2A) because it did not prevent tumor initiation, and the tumors grew progressively until essentially all the chickens had died. The differences among controls that died (80%) and those treated with R-line HMW-I that died (80%) and those treated with Pr-line HMW-I (100%) are not significant as determined by Student's t test (Davies, 1954). It can be concluded that anti-RSV
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DAYS POST INITIAL RSV INOCULATION FIG. 2. Preincubation of RSV with HMW-I (A) and LMW-II (B) followed by injection into Pr-line chicken wing-webs. HMW-I and LMW-II were isolated from R-line and Pr-line sera (obtained before RSV 8 9 10 II 12 13 14 15 16 17 challenge) by Sephadex G-100 column chromatography. In A, ( • ) represents treated group ELUTION VOLUME (ml) where each chicken received an injection containing x 100 RSV plus 120 mg HMW-I from R-line unsensitized chicken sera, (- - A - -) represents treated group where FIG. 1. Sephadex G-100 column chromatography each chicken received an injection containing RSV elution profile of R-line hyperimmune sera fractions plus 120 mg HMW-I from Pr-line unsensitized chicken and molecular weight standards. R-line hyperimmune sera in a sample volume of 125 ml was applied to sera and ( o ) represents control group where the column and the sera components with molecular each chicken received RSV only. In B, (- - -• ) weights greater than 14,000 that eluted between represents treated group where each Pr-chicken 200 and 700 ml were collectively pooled and~ conreceived an injection containing RSV plus 5 mg centrated (HMW-I); sera components with molecular LMW-II from R-line unsensitized chicken sera, weights of 14,000 or less that eluted between 800 ( - - * - - ) represents treated group where each chicken and 1500 ml were collectively pooled and concenreceived an injection containing RSV plus 5 mg trated (LMW-II). All other sera samples were treated LMW-II from Pr-line unsensitized chicken sera, and ( o ) represents control group where each similarly. Symbols: ( o ), elution profile chicken received RSV only. Numbers in bold face of R-line hyperimmune sera fractionation; ( * ), print represent the number of chickens remaining rechromatography of an a l i q u o t of LMW-II; alive at various times post initial RSV inoculation ( • ), profile of molecular weight standards in treated and control groups. Tumor scores decline (A, blue dextran; B, bovine hemoglobin; C, lysozyme; after sensitive chickens die. D, potassium ferricyanide).
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LMW-II components. Each fraction was tested for anti-RSV activity by incubation with RSV and subsequent injection into the wing-web of 8-week-old Pr-chickens. Tumors were scored (Gyles et al, 1977) periodically and the results of these experiments are recorded in Figures 2, 3, and 4. Average tumor score after 30 days declined because the susceptible Pr-chickens had died and the more resistant chickens had begun to regress their tumors. There is no evidence for virus neutralizing activity in either HMW I or LMW II from unsensitized chickens. The sizes of the group of chickens at the start and at various times during the experiment are given in boldface letters in the figures. In these experiments, HMW-I and LMW-II clearly protect Pr-chicken when the samples are isolated from sensitized animals.
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antibodies are not present in the HMW-I components of the sera in either the R- or Prflocks of unchallenged chickens.
CO
O
Figure 3A shows the effect of R-line HMW-I prepared from chicken sera 32 days postinoculation (after regression) and the effect of Pr-line HMW-I prepared from sera 20 days postinoculation. R-line HMW-I completely suppressed tumorgenesis of the RSV after
UJ
< UJ
4.03.0-
DAYS POST INITIAL RSV INOCULATION
FIG. 3. Preincubation of RSV with HMW-I (A) and LMW-II (B) followed by injection into Pr-line chicken wing-webs. HMW-I and LMW-II were isolated from R-line and Pr-line immune sera by Sephadex G-100 column chromatography. In A, (- - -•- - -) represents treated group where each chicken received an injection containing RSV plus 120 mg HMW-I from R-line immune sera obtained 2 weeks after tumor regression (32 days post RSV challenge), ( - - * - - ) represents treated group where each chicken received an injection containing RSV plus 120 mg HMW-I from Pr-line immune sera obtained while chickens were carrying #4 size tumors (20 days post RSV challenge) and ( o ) represents control group where each chicken received an injection containing RSV only. In B, ( • ) represents treated group where each chicken received an injection containing RSV plus 5 mg LMW-II from R-line immune sera obtained 32 days post RSV challenge, ( - - * - - ) represents treated group where each chicken received an injection containing RSV plus 5 mg LMW-II from Pr-line immune sera obtained 20 days post RSV challenge, and ( o ) represents control group where each chicken received RSV only. Numbers in boldface print as in Figure 2.
2.0 1.0 < ir
0-
DAYS POST INITIAL RSV INOCULATION
FIG. 4. Preincubation of RSV with HMW-I and LMW-II followed by injection in Pr-line chicken wing webs. HWM-I and LMW-II were isolated from R-line hyperimmune sera by Sephadex G-100 column chromatography. In the figure, ( • ) represents treated group where each chicken received an injection containing RSV plus 120 mg HMW-I, ( - - * - - ) represents treated group where each chicken received an injection containing RSV plus 5 mg LMW-II and ( o ) represents control group where each chicken received RSV only. Numbers in boldface print as in Figure 2.
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o o
Similarly, neither the R- nor Pr-line LMW-II from sera from unchallenged chickens exhibited any anti-RSV activity against a standard inoculum of RSV. The pertinent data are recorded in Figure 2B. In the group treated with RSV preincubated with R-line LMW-II, tumors were initiated and grew progressively to # 4 scores before regressing or showing definite signs of regression in 43% of 33 chickens. The remaining 57% treated died or had #4 scores 75 days postinjection. Thirty percent of the control chickens regressed tumors, which was not significantly different from the treated group. Figure 2B also shows that preincubation with RSV of Pr-line LMW-II from sera of unchallenged chickens does not inactivate the RSV. Seventy-eight percent of the treated chickens injected died and 22% regressed, and there was no significant difference between treated and control groups as judged by Student's t test (Davies, 1954).
CHICKEN SERA TO NEUTRALIZE RSV
As expected (see Fig. 4), the HMW-I from hyperimmune sera from R-line chickens was capable of completely arresting the activity of RSV. None of the injected chickens developed tumors, whereas 80% of the control group were dead or carrying # 4 tumors 32 days postinoculation (P<.01). Reimmunization of the R-chickens with RSTH (hyperimmune chickens) restimulates the production of anti RSV-LMW-II as shown in Figure 4. In the treated group, 35% of the chickens did not develop tumors, whereas die other 55% regressed or were regressing tumors during the 32 days observation period. At the end of the experiment 90% of the treated group had no tumors whereas 80% of the control group had died. The difference is significant
(P<.01). This is a very interesting result, because LMW-II components are not present in R-line chicken immune sera 32 days post-RSV (after tumor regression) but can be restimulated by a booster inoculation of RSV or tumor homogenate.
DISCUSSION
The most interesting aspect of this study was the observation of a low molecular weight component in both progressor and regressor sera that possessed viral neutralizing activity. The origin of the anti-RSV LMW-II component is unknown, whereas HMW-I is largely or exclusively immunoglobulin (Allen, 1978). It cannot be detected by immunoelectrophoresis with either anti-chicken IgM or IgG antisera. The LMW-II component disappeared 2 weeks after tumor regression in the R-chickens (32 days post-RSV challenge), reappeared upon subsequent rechallenge in the R-line chickens, and just began to appear in high levels in the Pr-chickens at 20 days after RSV challenge when the chickens were carrying large tumors. It is intriguing that cytotoxic T-cells apparently disappear during the regressive phase of tumor development, which makes them attractive candidates as LMW-II producers. The LMW-II and cytotoxic T cells' disappearance after regression can be restimulated by tumor homogenate and immunoglobulins titers remain high for several months (Fink and Rauscher, 1961). These observations also suggest that the LMW-II is not an immunoglobulin fragment. The LMW-II does not stain on SDS-polyacrylamide electrophoresis (data not shown). The partition coefficient of die LMW-II substance is unity on the Sephadex G-100 column so that we estimate its molecular weight is less than 5,000 daltons (Pharmacia Fine Chemicals, 1975). The mechanism of action of LMW-II on virus is not known, but it could be inactivating RSV from transformed cells (Wainberg et al., 1979). The possibility, of course, exists that the LMW-II has general antiviral activity and acts by inhibiting nucleic acid replication (Frume et al, 1979; Burzynski^a/., 1976). ACKNOWLEDGMENTS We thank Van Thompson, Omer McConnell, Olen Dunaway, and Paul Teague for their friendly and constant help at the University of
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preincubation. By 41 days postinoculation, none of the treated chickens had developed tumors, whereas 87% of the control animals had died (P<.01) from Rous sarcomas. The Pr-line HMW-I also had anti-RSV activity. Thirty percent of the animals challenged with RSV preincubated with Pr-line HMW-I developed small tumors, but all had completely regressed within 41 days after challenge and significantly differ from the controls (P<.01). The difference in tumor takes between the R-line HMW-I (0%) and the Pr-line HMW-I (30%) treated RSV is not significant but hints at a greater RSV neutralizing activity in the R-sera. The levels of anti-RSV activity in the LMWII from R-chickens taken at 32 days postinoculation and from Pr-chickens taken 20 days post-RSV challenge were unexpected (Fig. 3B). The R-line LMW-II activity had disappeared, because 80% of the treated chickens died (received RSV plus R-line LMW-II) with progressive tumors and only 20% showed signs of tumor regression. The treated group did not differ significantly from the control group where 67% had died and the rest were carrying #4 tumors at the termination of the experiment 79 days postchallenge. However, pretreatment of RSV with LMW-II from Pr-birds carrying heavy tumor burdens (score 4) was effective in reducing the infectivity of the virus. In the treated group, 56% of the chickens did not develop visible tumors and the remaining 44% either regressed tumors or were showing definite signs of tumor regression at the termination of the experiment. By day 79 postchallenge, 88% of the birds had no apparent tumor and differed from the control (P<.01).
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Arkansas poultry farrp. Without their help, this work would not have been possible. REFERENCES
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Allen, J., 1978. Studies on the Effect of Purified Rous Sarcoma Virus Specific Antibody on Rous Sarcoma Regression in Chickens. Univ. Arkansas, Fayetteville, AR. Burzynski, S. R., 1976. Antineoplastins: Biochemical defense against cancer. Physiol. Chem. Phys. 8:275-279. Collins, W. M., W. E. Briles, R. M. Zsigray, W. R. Dunlop, A. Corbett, K. K. Clark, J. R. Marks, and T. P. McGrail, 1977. The B locus (MHC) in the chicken: Association with the fate of RSVinduced tumors. Immunogenetics 5:333 — 343. Davies, O. L., 1954. Statistical Methods in Research and Production. Oliver and Boyd, London. Fink, M., and F. Rauscher, 1961. A simple method for potent chicken anti-Rous sarcoma virus serum. J. Natl. Cancer Inst. 26:519-522. Frume, L., A. Mattioli, and C. Busi, 1979. An endog e n o u s inhibitor of fibroblast proliferation hinders Simian virus 40 replication. Naturwissenschaften 66:265—266. Gyles, N. R., and C. J. Brown, 1971. Selection in chickens for retrogression of tumors caused by Rous sarcoma virus. Poultry Sci. 50:901—905.
Gyles, N. R., M. Blythe, P. Test, A. Bowanki, and C. J. Brown. 1977. Immune response in progressor and regressor strains of chickens at specific intervals after primary challenge with Rous sarcoma virus. Poultry Sci. 56:758-766. Gyles, N. R., J. L. Miley, and C. J. Brown, 1967. The response of resistance and susceptible strains of chickens and their F, and F 2 crosses to subcutaneous inoculations with Rous sarcoma virus. Poultry Sci. 46:465-472. Gyles, N. R., B. R. Stewart, and C. J. Brown, 1968. Mechanisms of genetic resistance in trie chicken to Rous sarcoma virus. Poultry Sci. 47:430-450. Lowry, D. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 1 9 3 : 2 6 5 275. Pharmacia Fine Chemicals. 1975. Sephadex gel filtration in theory and practice. Upplands Grafiska AB, Uppsala, Sweden. Schierman, L. W., D. H. Watanabe, and R. A. McBride, 1977. Genetic control of Rous sarcoma regression in chickens: Linkage with the major histocompatibility complex. Immunogenetics 5: .325-332. Wainberg, M. A., B. Buss, R. Wahi, and E. Israel, 1979. The thymic dependence of cell-mediated immunity to avian sarcomas in chickens. Cell Immunol. 45:344-355.