Proc. Nati. Acad. Sc. USA Vol. 76, No. 4, pp. 2018-2021, April 1979 Medical Sciences
Protective effect of immunization with nonviral antigens against Friend leukemia virus in mice aponAt oly(uimethylated bovine serum albumin/bovine serum albumin/dinitrophenylated keyhole limpet hemocyanin/gp7l Idy EVELYNE NAHON-MERLIN*, FANNY LACOUR*, CHARLOTTE FRIENDt, AND ROBERTO REVOLTELLAt ant
*Institut Gustave-Roussy, GR 8 du Centre National de la Recherche Scientifique, 94800 Villejuif, France; tMollie B. Roth Laboratory, Center for Experimental Cell Biology, Mt. Sinai School of Medicine of the City University of New York, New York, New York 10029; and tLaboratorio di Biologia Cellulare, Consiglio Nazionale delle Richerche, Rome, Italy 00196
Contributed by Charlotte Friend, January 8, 1979
ABSTRACT DBA/2 mice immunized with poly(A).poly(U) complexed with methylated bovine serum albumin and emulsified in Freund's complete adjuvant were protected against challenge with Friend leukemia virus. There was no correlation between the level of antibody to the immunogen in the prechallenge serum and induced resistance to the virus. Although prechallenge sera of mice given the same amount of the duplex in a single inoculum bound 9.7% of poly(A) poly([3HJU) input, as compared to 45.3% bound by the prechallenge sera of mice given the immunogen in divided doses, both groups of mice were equally resistant to infection. Immunization with two other nonviral agents, bovine serum albumin fraction V or dinitrophenylated keyhole limpet hemocyanin, induced the same level of protection. A sparing effect of approximately 1015 in infectivity was afforded the immunized mice. Immunization with either poly(A)poly(U) alone or with the carrier methylated bovine serum albumin was ineffective. In addition to antibodies to the respective immunogens, the prechallenge sera of the immunized mice also contained antibody to Friend leukemia virus gp7l. The presence of such viral infection by antiodies was not always related to resistance toinfection did Friend virus. Some immunized mice that survived not have gp7l antibody in their serum before challenge, and mice immunized with poly(A)'poly(U) alone were susceptible to infection, although their prechallenge sera containe antibody to gp7l. The mechanism involved in the induction of resistance to infection is not known. The effect may be mediated through a modification of the expression of both endogenous and exogenous type C viruses and affect immunological mechanisms controlling cellular responses. Stimulation with a given antigen elicits not only specific antibodies, but also a variety of other immunoglobulins (1-7). Some of these react with compounds totally unrelated to the immunogen (7-9). The mechanisms involved in this phenomenon are not understood, but it has been suggested that immunologic stimulation may be involved in the process of lymphocyte maturation (7). In order to explore the role of antigenic stimulation in the development of immunity to virus infection, we are studying the effect of immunization with nonviral agents on Friend leukemia virus (FLV), which induces a fulminating erythroleukemia after a short incubation period of 1-2 weeks in adult susceptible mice (10). We had previously shown that mice hyperimmunized with poly(I)-poly(C) complexed with methylated bovine serum albumin (MBSA) in complete Freund's adjuvant (CFA) developed antibodies to the duplex and were protected against FLV infection (11). The present report describes the protective effect against FLV of immunization with poly(A)-poly(U)-MBSA/CFA, bovine serum al-
bumin fraction V, [BSA(V)]/CFA, and dinitrophenylated keyhole limpet hemocyanin (DNP-KLH)/CFA. All were found to afford the same level of protection. Immunization with either the carrier MBSA/CFA or poly(A)-poly(U) alone was ineffective. Although the prechallenge sera of the immunized mice contained antibody that reacted with FLV gp71, the increase in such viral antibody was not always associated with protection against subsequent infection. MATERIALS AND METHODS Antigens. Preparation of poly(A), poly(U), and the complex has been described (12). The labeled double-strandedhelix was formed by mixing poly(A) (Boehringer Mannheim) and poly([5-3H]U) (Miles) in an equimolar ratio. The interaction between the two polymers was checked by measuring the hypoor hyperchromicity of the complex at different wavelengths (13). MBSA was purchased from Worthington and BSA(V) from Worthington and from Armour Co. (Chicago, IL). DNPm-KLH was kindly provided by Z. Ovary (New York University Medical Center, New York, NY). Immunization. Female DBA/2J and DBA/2 ORL mice, approximately 2 months old, were obtained from The Jackson Laboratory and CSEAL (Orleans, France). Poly(A)-poly(U) alone, poly(A)-poly(U)-MBSA/CFA, or MBSA/CFA were injected according to the schedule previously described (11). Briefly, a series of six inoculations, each containing 40 ,ug of the duplex with or without 40 ,ug of MBSA/CFA, or MBSA/CFA alone, were given. BSA(V), diluted in saline, was emulsified in CFA. One intraperitoneal injection of 100 ,g was followed by two injections (50 ,ug each) at weekly intervals. DNP-KLH was emulsified in CFA and two intraperitoneal injections (10 ,ug each) were given 1 month apart. Each of the immunized mice was bled retro-orbitally 1 week after the final inoculation of antigen and the sera were stored at -200 C. The day after the sera were collected, the mice were challenged with 10-fold dilutions of FLV prepared from filtrates of leukemic spleens as described (10). The experiments were terminated after 6 months. In parallel experiments, encephalomyocarditis virus, kindly provided by M. L. Giron (Inst. Gustave-Roussy, Villejuif, France) was used to challenge mice that had been immunized with BSA(V). Detection of Antibodies. The radioimmunoassay used to
Abbreviations: BSA(V), bovine serum albumin fraction V; MBSA, methylated bovine serum albumin; CFA, complete Freund's adjuvant; albuThe publication costs of this article were defrayed in part by page DNPW-BSA and DNP29KLH, dinitrophenylated bovine serumrefer to min and keyhole limpet hemocyanin, respectively (subscripts charge payment. This article must therefore be hereby marked "adthe number of DNP groups per 105 daltons for KLH and per molecule vertisement" in accordance with 18 U. S. C. §1734 solely to indicate of protein for BSA); FLV, Friend leukemia virus. this fact. 2018
Medical Sciences: Nahon-Merlin et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
2019
Table 1. Effect of immunization with poly(A).poly(U)-MBSA/CFA on FLV infection in mice Virus dilution*
10-1
10-2
10-3
10-4
LD5ot
7/7
7/7
7/7
0/7
3.5
4/6
2/7
3/7
0/7
1.8
7/7
6/6
5/7
4/7
3.9
1/7 7/7 5/6 (single injection) *Values are no. of leukemic mice/no. inoculated. t Mean lethal dose. t Prechallenge serum.
0/7
2.5
DBA/2J mice
log
% immunogen
difference
bound*t
Experiment 1
Control
2.5
Immunized
(multiple injections) Experiment 2 Control Immunized
detect the presence of antibody against poly(A)-poly(U) was a modification of the Farr technique (14). Reaction mixtures of 150 til were prepared by adding poly(A)-poly([5-3H]U) (0.2 ,ug, 8600 cpm) to 10 1.l of decomplemented serum in Trisbuffered saline (pH 7.5). The tubes were incubated at 370C for 1 hr and 4VC for 16 hr. After addition of 600 ,.g of immunoglobulin, a volume of cold 70% saturated ammonium sulfate equal to that of the reaction mixture was added. The precipitate was allowed to form for 1 hr at 4°C, and the antigen-antibody complex was separated from unbound poly(A)-poly([3H]U) by centrifugation. The precipitates were washed three times with 35% saturated ammonium sulfate and solubilized in Soluene (Packard, Downers Grove, IL). After dispersion in 10 ml of scintillation liquid, radioactivity was measured in duplicate samples. Less than 5% of the labeled antigen coprecipitated with the globulin from control normal mouse sera. The results are expressed as percentages of radioactivity recovered in the precipitate as compared to the total input. Radioimmunoassay used for detecting FLV gp7l antibodies was a modification of the Farr technique (14) described previously (15). FLV gp7l and goat anti-FLV gp7l were kindly provided by Dani Bolognesi (Duke University, Durham, NC). Tyrosine residues in gp71 were labeled with 125I by the chloramine T iodination procedure (16). Rabbit anti-mouse gamma globulin serum was used to precipitate the antigen-antibody complex. The total mass of antibody was calculated from the specific activity of the labeled antigen (cpm per ng of 125Ilabeled antigen precipitated by a hyperimmune serum, usually 90% of the input) by the following equation: 52I-labeled antigen bound in test cpm precipitated in test sample blank specific activity of antigen -
1.7
45.3
1.4
9.7
Titration of anti-DNP-KLH antibody of individual sera were determined by hemagglutination with tannic acid-treated DNP66-BSA-coated erythrocytes (17).
RESULTS Effect of immunization with nonviral agents Each of the test immunogens was administered according to the protocols described in Materials and Methods. Sera collected from the immunized mice 1 day before challenge with FLV were assayed for antibody content. The first series of experiments were carried out with poly(A).poly(U). It was of interest to examine this duplex since immunization with poly(I).poly(C) had provided a sparing effect against FLV infection (11). The binding activity of 10 ,ul of the prechallenge sera of mice inoculated with six doses of poly(A)-poly(U)-MBSA/CFA to poly(A)-poly([3H]U) was 45.3% as compared to 2.5% bound by the sera of normal mice of the same age. After challenge with FLV, the protective effect of immunization was detected at each dilution of the virus (Table 1, Exp. 1). There was a 1.7 log difference in ineffectivity between the untreated and immunized mice. The immunized survivors remained free of disease for the 6-month observation period. To determine whether the binding capacity of the antibodies was associated with the degree of protection against the virus, we performed the next experiment in mice given a single intraperitoneal injection of 250 ,tg of poly(A)-poly(U) and 250 ,ug of MBSA in CFA (Table 1, Exp. 2). The prechallenge sera of these mice bound only 9.7% of the poly(A)-poly([3H]U) input compared to 45% bound by sera of mice immunized with the same amount of immunogen given in divided doses. Never-
Table 2. Effect of multiple injections of poly(A)-poly(U) or MBSA/CFA on FLV infection of mice
DBA/2J mice
10-'
Virus dilution* 10-3 10-2
Experiment 1 Control Poly(A)-poly(U)
10/10 10/10
10/10 10/10
Experiment 2 Control
7/7 6/6 7/7 7/7 * Values are no. of leukemic mice/no. inoculated.
MBSA/CFA
t Mean lethal dose. *
Prechallenge serum. ND, not done.
% immunogen
10-4
LD50t
boundt
10/10 9/10
6/10 6/10
>4 >4
2.0
7/7 7/7
1/7 1/8
3.6 3.6
ND
2020
Medical Sciences: Nahon-Merlin et al.
Proc. Nati. Acad. Sci. USA 76 (1979)
Table 3. Effect of BSA(V)/CFA immunization against FLV infection in mice Virus dilution* log 10-1 10-2 10-4 10-3 DBA/2J mice LD5ot difference Experiment 1 9/10 >4 10/10 Control 7/9 10/10 NDt 3/10 2.8 5/10 8/9 6/10 Immunized Experiment 2 ControlImmunized
10/10 8/9
10/10 4/8 *Values are no. of leukemic mice/no. inoculated.
9/9 3/10
3/9 0/8
3.8 2.2
1.6
t Mean lethal dose. t Not done.
theless, the sparing effect against the virus was comparable. The log difference in infectivity between control and immunized mice was 1.4 and 1.7, respectively. These data indicate that high antigen-binding capacity is not necessarily associated with protection against FLV. Control experiments
were then carried out to check if poly(A)-poly(U) alone or MBSA/CFA could elicit a protective effect. Series of six inoculations of poly(A)-poly(U) without carrier were ineffective against FLV. The prechallenge sera bound 2%' of poly(A)-poly([3H]U), approximately the level found in control unimmunized mice. Similar results were obtained with mice immunized with MBSA/CFA. There was no difference in the ability of the control or the immunized mice to resist infection (Table 2). The possibility that the negative results obtained with MBSA could be due to altered antigenicity was considered in view of the report that the ability of BSA to induce antibodies decreased when carboxyl groups were esterified by methyl alcohol (18). Therefore, the effect of immunization with the highly antigenic protein BSA(V) emulsified in CFA was studied. The results obtained with two different preparations are shown in Table 3. Antibodies specific for BSA(V) assayed by gel immunodiffusion were present in the prechallenge sera collected from immunized mice in both
experiments. The sparing effect afforded to mice immunized with BSA(V)/CFA was comparable to that observed in mice immunized with poly(A)-poly(U)-MBSA/CFA. Immunization with BSA(V) was not effective against ence-
phalomyocarditis virus (data not shown), confirming our previous findings that immunization with poly(I)-poly(C)MBSA/CFA afforded no protection against this lytic virus (11).
The observation that lymphocytes of mice antigenically stimulated either in vivo or in Aitro by DNP-KLH displayed the major FLV envelope glycoprotein gp7l on their surfaces (19) prompted us to examine the effect of immunization with this agent. The prechallenge sera of 9 of the 40 immunized mice were assayed individually for antibody to the immunogen. Each serum agglutinated tannic acid-treated DNP-BSA-coated sheep erythrocytes at a final dilution ranging from 1/64 to 1/256. The Table 4. Effect of DNP-KLH/CFA immunization on FLV infection in mice Virus dilution*
*
DBA/2J mice
10-1
10-2
10-3
10-4
LD50t
Control Immunized
10/10 10/10
10/10 5/10
10/10 4/10
8/10 0/10
>4
Values are no. of leukemic mice/no. inoculated.
t Mean lethal dose.
2.4
protection afforded to mice immunized with DNP-KLH was
of the same order of magnitude as had been obtained after immunization with poly(A)-poly(U)-MBSA/CFA and BSA(V)/CFA (Table 4). Detection of antibodies to FLV gp7l In order to determine whether or not the activation of gp71 was involved in the protective effect induced by the nonviral agents, we assayed the prechallenge serum of individual mice immunized with either poly(A)-poly(U)-MBSA/CFA or DNP-KLH for the presence of antibodies to FLV gp7l. Each of the five mice immunized with poly(A)-poly(U)-MBSA/CFA and four of the five mice immunized with DNP-KLH had antibodies that bound to 125I-labeled FLV gp7l (Table 5). Viral gp71 antibodies were also detected in the prechallenge sera of mice immunized with poly(A)-poly(U) alone, which had been ineffective in protecting against the virus. The results are summarized in Table 6. DISCUSSION A significant level of protection against FLV infection was induced in mice antigenically stimulated with nonviral immunogens. The innoculation of poly(A)-poly(U)-MBSA/CFA, BSA(V)/CFA, or DNP-KLH/CFA elicited antibodies to the respective immunogens. The immunized mice were able to withstand infection with approximately 1.5 log units more FLV than the untreated controls. However, there appeared to be no correlation between the antibody response to the immunogen in the prechallenge serum and the subsequent resistance to FLV. This is evident in comparing the results obtained after immunization with the same amount of poly(A)-poly(U)MBSA/CFA given in a single inoculation or in divided doses (Table 1). Although there was less than 25% of the binding activity to the labeled immunogen in the sera of mice immunized with a single injection, both groups were equally resistant to challenge with the virus. Table 5. Binding capacity of individual prechallenge sera from immunized mice to 125I-labeled FLV gp7l Serum from Serum from mice immunized with unimmunized Poly(A)-poly(U)DNP-KLH control MBSA/CFA 8.30 14.20 0.80 114.12 0.14 14.20 41.4 0.10 1.20 4.0 15.2 12.14 1.00 0.10 Total mass of antibody was determined by antigen-binding capacity and is expressed as ,ug/antibody per ml. The binding capacity of goat anti-gp7l was 214.6.
Medical Sciences: Nahon-Merlin et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
Table 6. Summary of results Antibodies (prechallenge serum) ProtecImmution Treatment nogen gp7l 2.5* None Poly(A)-poly(U)-MBSA/CFA + + 45.3* (multiple injections) ND 2.5* None Poly(A)-poly(U)-MBSA/CFA + ND 9.7* (single injection) 2.5* None + 2.0* Poly(A)-poly(U) ND ND None ND MBSA/CFA None
_t
BSA(V)/CFA
+t
None BSA(V)/CFA
_t
None DNP-KLH
--
+t
ND + ND + +
+
+ +
ND, not done. * % poly(A)-poly([3H]U) bound per 10,gl of serum.
t Qualitative gel immunodiffusion.
Tannic acid-treated DNP-BSA-coated sheep erythrocyte hemagglutination.
It has been suggested that the expression of gp7l and of en-
dogenous type C virus synthesis after nonspecific immunization may signal specific events in the differentiation of the stimulated lymphocytes (19). However, the role of the antibodies to gp71 elicited by the immunogens in the present study is paradoxical. They are rarely present in normal, untreated mice of the same age, yet their appearance in the immunized mice was not always indicative of subsequent resistance to FLV. The prechallenge serum of some of the mice surviving infection had not contained antibody to FLV gp71. On the other hand, mice immunized with poly(A)-poly(U) alone were susceptible to infection, although their prechallenge serum contained antibody to gp7l. Thus, the induction of these antibodies may merely be a signal of the activation of essential immunological mechanisms which include those controlling cellular responses. This is suggested by the fact that approximately the same degree of protection was afforded by each of the effective nonviral, unrelated immunogens. Results comparable to ours have been obtained in mice immunized with FLV gp71/CFA (20). In that study, the development of splenomegaly was inhibited in immunized mice challenged with t103 plaque-forming units of FLV. The level of protection achieved after immunization
2021
with gp71 (20) or with the nonspecific immunogens described here is of a lower order than that obtained after immunization with whole virions (21). Our results indicate that immunization with nonviral agents against retroviruses may be promising, although the mechanisms involved in the induction of resistance are not known. Interferon does not appear to play a role since it was not detectable in serum at the time the immunized mice were challenged with FLV (11). The protective effect may be mediated through a modification of both endogenous and exogenous type C virus expression. We thank Dr. Zoltan Ovary for helpful discussions and for the DNP-KLH antibody titrations performed in his laboratory. We thank Colette Chianale and J. Gilbert Holland for excellent technical assistance and Loris Harrington for expert secretarial assistance. This work was supported in part by National Cancer Institute Grants CA 10,000 and CA 13,047. 1. Urbain-Vansanten, G. (1970) Immunology 19,783-797. 2. Antoine, J. C. & Avrameas, S. (1973) Ann. Immunol. (Paris) 124C, 121-131. 3. Miller, H. R. P., Ternynck, T. & Avrameas, S. (1974) Ann. Immunol. (Paris) 125C, 231-238. 4. Moticka, E. J. (1974) Immunology 27,401-412. 5. Pachmann, K., Killander, D. & Wigzell, H. (1974) Eur. J. Immunol. 4, 138-145. 6. Miller, H. R. P., Ternynck, T. & Avrameas, S. (1975) J. Immunol. 114,626-629. 7. Poskitt, D. C., Frost, H., Cahill, R. N. P. & Trnka, Z. (1977) Immunology 33,81-89. 8. Cameron, D. J. & Erlanger, B. F. (1977) Nature (London) 268, 763-765. 9. Richards, F. F., Konigsberg, W. H., Rosenstein, R. W. & Varga, J. M. (1975) Science 187, 130-137. 10. Friend, C. (1957) J. Exp. Med. 105,307-318. 11. Fourcade, A., Friend, C., Lacour, F. & Holland, J. G. (1974) Cancer Res. 34, 1749-1751. 12. Fenster, E. D., Lacour, F. & Harel, J. (1975) Exp. Cell Res. 94,
315-320. 13. Michelson, A. M., Massoulie, J. & Guschlbauer, W. (1967) Prog. Nucleic Acid Res. Mol. Biol. 6,83-141. 14. Farr, R. S. (1958) J. Infect. Dis. 103, 239-262. 15. Revoltella, R., Adler, L. T. & Osler, A. G. (1971) J. Immunol. 106, 1507-1518. 16. Greenwood, F. C., Hunter, W. M. & Glover, J. S. (1963) Biochem. J. 89, 114-123. 17. Stavitsky, A. B. (1954) J. Immunol. 72,360-367. 18. Maret, A. & Drach, G. (1974) C. R. Hebd. Seances Acad. Sci. Ser. D 278,2019-2022. 19. Wecker, E., Schimpl, A. & Huinig, T. (1977) Nature (London)
269,598-600. 20. Hunsmann, G., Moennig, V. & Schafer, W. (1975) Virology 66,
327-329. 21. Friend, C. & Rossi, G. B. (1968) Int. J. Cancer 3,523-529.
Protective effect of immunization with nonviral antigens against Friend leukemia virus in mice aponAt oly(uimethylated bovine serum albumin/bovine serum albumin/dinitrophenylated keyhole limpet hemocyanin/gp7l Idy EVELYNE NAHON-MERLIN*, FANNY LACOUR*, CHARLOTTE FRIENDt, AND ROBERTO REVOLTELLAt ant
*Institut Gustave-Roussy, GR 8 du Centre National de la Recherche Scientifique, 94800 Villejuif, France; tMollie B. Roth Laboratory, Center for Experimental Cell Biology, Mt. Sinai School of Medicine of the City University of New York, New York, New York 10029; and tLaboratorio di Biologia Cellulare, Consiglio Nazionale delle Richerche, Rome, Italy 00196
Contributed by Charlotte Friend, January 8, 1979
ABSTRACT DBA/2 mice immunized with poly(A).poly(U) complexed with methylated bovine serum albumin and emulsified in Freund's complete adjuvant were protected against challenge with Friend leukemia virus. There was no correlation between the level of antibody to the immunogen in the prechallenge serum and induced resistance to the virus. Although prechallenge sera of mice given the same amount of the duplex in a single inoculum bound 9.7% of poly(A) poly([3HJU) input, as compared to 45.3% bound by the prechallenge sera of mice given the immunogen in divided doses, both groups of mice were equally resistant to infection. Immunization with two other nonviral agents, bovine serum albumin fraction V or dinitrophenylated keyhole limpet hemocyanin, induced the same level of protection. A sparing effect of approximately 1015 in infectivity was afforded the immunized mice. Immunization with either poly(A)poly(U) alone or with the carrier methylated bovine serum albumin was ineffective. In addition to antibodies to the respective immunogens, the prechallenge sera of the immunized mice also contained antibody to Friend leukemia virus gp7l. The presence of such viral infection by antiodies was not always related to resistance toinfection did Friend virus. Some immunized mice that survived not have gp7l antibody in their serum before challenge, and mice immunized with poly(A)'poly(U) alone were susceptible to infection, although their prechallenge sera containe antibody to gp7l. The mechanism involved in the induction of resistance to infection is not known. The effect may be mediated through a modification of the expression of both endogenous and exogenous type C viruses and affect immunological mechanisms controlling cellular responses. Stimulation with a given antigen elicits not only specific antibodies, but also a variety of other immunoglobulins (1-7). Some of these react with compounds totally unrelated to the immunogen (7-9). The mechanisms involved in this phenomenon are not understood, but it has been suggested that immunologic stimulation may be involved in the process of lymphocyte maturation (7). In order to explore the role of antigenic stimulation in the development of immunity to virus infection, we are studying the effect of immunization with nonviral agents on Friend leukemia virus (FLV), which induces a fulminating erythroleukemia after a short incubation period of 1-2 weeks in adult susceptible mice (10). We had previously shown that mice hyperimmunized with poly(I)-poly(C) complexed with methylated bovine serum albumin (MBSA) in complete Freund's adjuvant (CFA) developed antibodies to the duplex and were protected against FLV infection (11). The present report describes the protective effect against FLV of immunization with poly(A)-poly(U)-MBSA/CFA, bovine serum al-
bumin fraction V, [BSA(V)]/CFA, and dinitrophenylated keyhole limpet hemocyanin (DNP-KLH)/CFA. All were found to afford the same level of protection. Immunization with either the carrier MBSA/CFA or poly(A)-poly(U) alone was ineffective. Although the prechallenge sera of the immunized mice contained antibody that reacted with FLV gp71, the increase in such viral antibody was not always associated with protection against subsequent infection. MATERIALS AND METHODS Antigens. Preparation of poly(A), poly(U), and the complex has been described (12). The labeled double-strandedhelix was formed by mixing poly(A) (Boehringer Mannheim) and poly([5-3H]U) (Miles) in an equimolar ratio. The interaction between the two polymers was checked by measuring the hypoor hyperchromicity of the complex at different wavelengths (13). MBSA was purchased from Worthington and BSA(V) from Worthington and from Armour Co. (Chicago, IL). DNPm-KLH was kindly provided by Z. Ovary (New York University Medical Center, New York, NY). Immunization. Female DBA/2J and DBA/2 ORL mice, approximately 2 months old, were obtained from The Jackson Laboratory and CSEAL (Orleans, France). Poly(A)-poly(U) alone, poly(A)-poly(U)-MBSA/CFA, or MBSA/CFA were injected according to the schedule previously described (11). Briefly, a series of six inoculations, each containing 40 ,ug of the duplex with or without 40 ,ug of MBSA/CFA, or MBSA/CFA alone, were given. BSA(V), diluted in saline, was emulsified in CFA. One intraperitoneal injection of 100 ,g was followed by two injections (50 ,ug each) at weekly intervals. DNP-KLH was emulsified in CFA and two intraperitoneal injections (10 ,ug each) were given 1 month apart. Each of the immunized mice was bled retro-orbitally 1 week after the final inoculation of antigen and the sera were stored at -200 C. The day after the sera were collected, the mice were challenged with 10-fold dilutions of FLV prepared from filtrates of leukemic spleens as described (10). The experiments were terminated after 6 months. In parallel experiments, encephalomyocarditis virus, kindly provided by M. L. Giron (Inst. Gustave-Roussy, Villejuif, France) was used to challenge mice that had been immunized with BSA(V). Detection of Antibodies. The radioimmunoassay used to
Abbreviations: BSA(V), bovine serum albumin fraction V; MBSA, methylated bovine serum albumin; CFA, complete Freund's adjuvant; albuThe publication costs of this article were defrayed in part by page DNPW-BSA and DNP29KLH, dinitrophenylated bovine serumrefer to min and keyhole limpet hemocyanin, respectively (subscripts charge payment. This article must therefore be hereby marked "adthe number of DNP groups per 105 daltons for KLH and per molecule vertisement" in accordance with 18 U. S. C. §1734 solely to indicate of protein for BSA); FLV, Friend leukemia virus. this fact. 2018
Medical Sciences: Nahon-Merlin et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
2019
Table 1. Effect of immunization with poly(A).poly(U)-MBSA/CFA on FLV infection in mice Virus dilution*
10-1
10-2
10-3
10-4
LD5ot
7/7
7/7
7/7
0/7
3.5
4/6
2/7
3/7
0/7
1.8
7/7
6/6
5/7
4/7
3.9
1/7 7/7 5/6 (single injection) *Values are no. of leukemic mice/no. inoculated. t Mean lethal dose. t Prechallenge serum.
0/7
2.5
DBA/2J mice
log
% immunogen
difference
bound*t
Experiment 1
Control
2.5
Immunized
(multiple injections) Experiment 2 Control Immunized
detect the presence of antibody against poly(A)-poly(U) was a modification of the Farr technique (14). Reaction mixtures of 150 til were prepared by adding poly(A)-poly([5-3H]U) (0.2 ,ug, 8600 cpm) to 10 1.l of decomplemented serum in Trisbuffered saline (pH 7.5). The tubes were incubated at 370C for 1 hr and 4VC for 16 hr. After addition of 600 ,.g of immunoglobulin, a volume of cold 70% saturated ammonium sulfate equal to that of the reaction mixture was added. The precipitate was allowed to form for 1 hr at 4°C, and the antigen-antibody complex was separated from unbound poly(A)-poly([3H]U) by centrifugation. The precipitates were washed three times with 35% saturated ammonium sulfate and solubilized in Soluene (Packard, Downers Grove, IL). After dispersion in 10 ml of scintillation liquid, radioactivity was measured in duplicate samples. Less than 5% of the labeled antigen coprecipitated with the globulin from control normal mouse sera. The results are expressed as percentages of radioactivity recovered in the precipitate as compared to the total input. Radioimmunoassay used for detecting FLV gp7l antibodies was a modification of the Farr technique (14) described previously (15). FLV gp7l and goat anti-FLV gp7l were kindly provided by Dani Bolognesi (Duke University, Durham, NC). Tyrosine residues in gp71 were labeled with 125I by the chloramine T iodination procedure (16). Rabbit anti-mouse gamma globulin serum was used to precipitate the antigen-antibody complex. The total mass of antibody was calculated from the specific activity of the labeled antigen (cpm per ng of 125Ilabeled antigen precipitated by a hyperimmune serum, usually 90% of the input) by the following equation: 52I-labeled antigen bound in test cpm precipitated in test sample blank specific activity of antigen -
1.7
45.3
1.4
9.7
Titration of anti-DNP-KLH antibody of individual sera were determined by hemagglutination with tannic acid-treated DNP66-BSA-coated erythrocytes (17).
RESULTS Effect of immunization with nonviral agents Each of the test immunogens was administered according to the protocols described in Materials and Methods. Sera collected from the immunized mice 1 day before challenge with FLV were assayed for antibody content. The first series of experiments were carried out with poly(A).poly(U). It was of interest to examine this duplex since immunization with poly(I).poly(C) had provided a sparing effect against FLV infection (11). The binding activity of 10 ,ul of the prechallenge sera of mice inoculated with six doses of poly(A)-poly(U)-MBSA/CFA to poly(A)-poly([3H]U) was 45.3% as compared to 2.5% bound by the sera of normal mice of the same age. After challenge with FLV, the protective effect of immunization was detected at each dilution of the virus (Table 1, Exp. 1). There was a 1.7 log difference in ineffectivity between the untreated and immunized mice. The immunized survivors remained free of disease for the 6-month observation period. To determine whether the binding capacity of the antibodies was associated with the degree of protection against the virus, we performed the next experiment in mice given a single intraperitoneal injection of 250 ,tg of poly(A)-poly(U) and 250 ,ug of MBSA in CFA (Table 1, Exp. 2). The prechallenge sera of these mice bound only 9.7% of the poly(A)-poly([3H]U) input compared to 45% bound by sera of mice immunized with the same amount of immunogen given in divided doses. Never-
Table 2. Effect of multiple injections of poly(A)-poly(U) or MBSA/CFA on FLV infection of mice
DBA/2J mice
10-'
Virus dilution* 10-3 10-2
Experiment 1 Control Poly(A)-poly(U)
10/10 10/10
10/10 10/10
Experiment 2 Control
7/7 6/6 7/7 7/7 * Values are no. of leukemic mice/no. inoculated.
MBSA/CFA
t Mean lethal dose. *
Prechallenge serum. ND, not done.
% immunogen
10-4
LD50t
boundt
10/10 9/10
6/10 6/10
>4 >4
2.0
7/7 7/7
1/7 1/8
3.6 3.6
ND
2020
Medical Sciences: Nahon-Merlin et al.
Proc. Nati. Acad. Sci. USA 76 (1979)
Table 3. Effect of BSA(V)/CFA immunization against FLV infection in mice Virus dilution* log 10-1 10-2 10-4 10-3 DBA/2J mice LD5ot difference Experiment 1 9/10 >4 10/10 Control 7/9 10/10 NDt 3/10 2.8 5/10 8/9 6/10 Immunized Experiment 2 ControlImmunized
10/10 8/9
10/10 4/8 *Values are no. of leukemic mice/no. inoculated.
9/9 3/10
3/9 0/8
3.8 2.2
1.6
t Mean lethal dose. t Not done.
theless, the sparing effect against the virus was comparable. The log difference in infectivity between control and immunized mice was 1.4 and 1.7, respectively. These data indicate that high antigen-binding capacity is not necessarily associated with protection against FLV. Control experiments
were then carried out to check if poly(A)-poly(U) alone or MBSA/CFA could elicit a protective effect. Series of six inoculations of poly(A)-poly(U) without carrier were ineffective against FLV. The prechallenge sera bound 2%' of poly(A)-poly([3H]U), approximately the level found in control unimmunized mice. Similar results were obtained with mice immunized with MBSA/CFA. There was no difference in the ability of the control or the immunized mice to resist infection (Table 2). The possibility that the negative results obtained with MBSA could be due to altered antigenicity was considered in view of the report that the ability of BSA to induce antibodies decreased when carboxyl groups were esterified by methyl alcohol (18). Therefore, the effect of immunization with the highly antigenic protein BSA(V) emulsified in CFA was studied. The results obtained with two different preparations are shown in Table 3. Antibodies specific for BSA(V) assayed by gel immunodiffusion were present in the prechallenge sera collected from immunized mice in both
experiments. The sparing effect afforded to mice immunized with BSA(V)/CFA was comparable to that observed in mice immunized with poly(A)-poly(U)-MBSA/CFA. Immunization with BSA(V) was not effective against ence-
phalomyocarditis virus (data not shown), confirming our previous findings that immunization with poly(I)-poly(C)MBSA/CFA afforded no protection against this lytic virus (11).
The observation that lymphocytes of mice antigenically stimulated either in vivo or in Aitro by DNP-KLH displayed the major FLV envelope glycoprotein gp7l on their surfaces (19) prompted us to examine the effect of immunization with this agent. The prechallenge sera of 9 of the 40 immunized mice were assayed individually for antibody to the immunogen. Each serum agglutinated tannic acid-treated DNP-BSA-coated sheep erythrocytes at a final dilution ranging from 1/64 to 1/256. The Table 4. Effect of DNP-KLH/CFA immunization on FLV infection in mice Virus dilution*
*
DBA/2J mice
10-1
10-2
10-3
10-4
LD50t
Control Immunized
10/10 10/10
10/10 5/10
10/10 4/10
8/10 0/10
>4
Values are no. of leukemic mice/no. inoculated.
t Mean lethal dose.
2.4
protection afforded to mice immunized with DNP-KLH was
of the same order of magnitude as had been obtained after immunization with poly(A)-poly(U)-MBSA/CFA and BSA(V)/CFA (Table 4). Detection of antibodies to FLV gp7l In order to determine whether or not the activation of gp71 was involved in the protective effect induced by the nonviral agents, we assayed the prechallenge serum of individual mice immunized with either poly(A)-poly(U)-MBSA/CFA or DNP-KLH for the presence of antibodies to FLV gp7l. Each of the five mice immunized with poly(A)-poly(U)-MBSA/CFA and four of the five mice immunized with DNP-KLH had antibodies that bound to 125I-labeled FLV gp7l (Table 5). Viral gp71 antibodies were also detected in the prechallenge sera of mice immunized with poly(A)-poly(U) alone, which had been ineffective in protecting against the virus. The results are summarized in Table 6. DISCUSSION A significant level of protection against FLV infection was induced in mice antigenically stimulated with nonviral immunogens. The innoculation of poly(A)-poly(U)-MBSA/CFA, BSA(V)/CFA, or DNP-KLH/CFA elicited antibodies to the respective immunogens. The immunized mice were able to withstand infection with approximately 1.5 log units more FLV than the untreated controls. However, there appeared to be no correlation between the antibody response to the immunogen in the prechallenge serum and the subsequent resistance to FLV. This is evident in comparing the results obtained after immunization with the same amount of poly(A)-poly(U)MBSA/CFA given in a single inoculation or in divided doses (Table 1). Although there was less than 25% of the binding activity to the labeled immunogen in the sera of mice immunized with a single injection, both groups were equally resistant to challenge with the virus. Table 5. Binding capacity of individual prechallenge sera from immunized mice to 125I-labeled FLV gp7l Serum from Serum from mice immunized with unimmunized Poly(A)-poly(U)DNP-KLH control MBSA/CFA 8.30 14.20 0.80 114.12 0.14 14.20 41.4 0.10 1.20 4.0 15.2 12.14 1.00 0.10 Total mass of antibody was determined by antigen-binding capacity and is expressed as ,ug/antibody per ml. The binding capacity of goat anti-gp7l was 214.6.
Medical Sciences: Nahon-Merlin et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
Table 6. Summary of results Antibodies (prechallenge serum) ProtecImmution Treatment nogen gp7l 2.5* None Poly(A)-poly(U)-MBSA/CFA + + 45.3* (multiple injections) ND 2.5* None Poly(A)-poly(U)-MBSA/CFA + ND 9.7* (single injection) 2.5* None + 2.0* Poly(A)-poly(U) ND ND None ND MBSA/CFA None
_t
BSA(V)/CFA
+t
None BSA(V)/CFA
_t
None DNP-KLH
--
+t
ND + ND + +
+
+ +
ND, not done. * % poly(A)-poly([3H]U) bound per 10,gl of serum.
t Qualitative gel immunodiffusion.
Tannic acid-treated DNP-BSA-coated sheep erythrocyte hemagglutination.
It has been suggested that the expression of gp7l and of en-
dogenous type C virus synthesis after nonspecific immunization may signal specific events in the differentiation of the stimulated lymphocytes (19). However, the role of the antibodies to gp71 elicited by the immunogens in the present study is paradoxical. They are rarely present in normal, untreated mice of the same age, yet their appearance in the immunized mice was not always indicative of subsequent resistance to FLV. The prechallenge serum of some of the mice surviving infection had not contained antibody to FLV gp71. On the other hand, mice immunized with poly(A)-poly(U) alone were susceptible to infection, although their prechallenge serum contained antibody to gp7l. Thus, the induction of these antibodies may merely be a signal of the activation of essential immunological mechanisms which include those controlling cellular responses. This is suggested by the fact that approximately the same degree of protection was afforded by each of the effective nonviral, unrelated immunogens. Results comparable to ours have been obtained in mice immunized with FLV gp71/CFA (20). In that study, the development of splenomegaly was inhibited in immunized mice challenged with t103 plaque-forming units of FLV. The level of protection achieved after immunization
2021
with gp71 (20) or with the nonspecific immunogens described here is of a lower order than that obtained after immunization with whole virions (21). Our results indicate that immunization with nonviral agents against retroviruses may be promising, although the mechanisms involved in the induction of resistance are not known. Interferon does not appear to play a role since it was not detectable in serum at the time the immunized mice were challenged with FLV (11). The protective effect may be mediated through a modification of both endogenous and exogenous type C virus expression. We thank Dr. Zoltan Ovary for helpful discussions and for the DNP-KLH antibody titrations performed in his laboratory. We thank Colette Chianale and J. Gilbert Holland for excellent technical assistance and Loris Harrington for expert secretarial assistance. This work was supported in part by National Cancer Institute Grants CA 10,000 and CA 13,047. 1. Urbain-Vansanten, G. (1970) Immunology 19,783-797. 2. Antoine, J. C. & Avrameas, S. (1973) Ann. Immunol. (Paris) 124C, 121-131. 3. Miller, H. R. P., Ternynck, T. & Avrameas, S. (1974) Ann. Immunol. (Paris) 125C, 231-238. 4. Moticka, E. J. (1974) Immunology 27,401-412. 5. Pachmann, K., Killander, D. & Wigzell, H. (1974) Eur. J. Immunol. 4, 138-145. 6. Miller, H. R. P., Ternynck, T. & Avrameas, S. (1975) J. Immunol. 114,626-629. 7. Poskitt, D. C., Frost, H., Cahill, R. N. P. & Trnka, Z. (1977) Immunology 33,81-89. 8. Cameron, D. J. & Erlanger, B. F. (1977) Nature (London) 268, 763-765. 9. Richards, F. F., Konigsberg, W. H., Rosenstein, R. W. & Varga, J. M. (1975) Science 187, 130-137. 10. Friend, C. (1957) J. Exp. Med. 105,307-318. 11. Fourcade, A., Friend, C., Lacour, F. & Holland, J. G. (1974) Cancer Res. 34, 1749-1751. 12. Fenster, E. D., Lacour, F. & Harel, J. (1975) Exp. Cell Res. 94,
315-320. 13. Michelson, A. M., Massoulie, J. & Guschlbauer, W. (1967) Prog. Nucleic Acid Res. Mol. Biol. 6,83-141. 14. Farr, R. S. (1958) J. Infect. Dis. 103, 239-262. 15. Revoltella, R., Adler, L. T. & Osler, A. G. (1971) J. Immunol. 106, 1507-1518. 16. Greenwood, F. C., Hunter, W. M. & Glover, J. S. (1963) Biochem. J. 89, 114-123. 17. Stavitsky, A. B. (1954) J. Immunol. 72,360-367. 18. Maret, A. & Drach, G. (1974) C. R. Hebd. Seances Acad. Sci. Ser. D 278,2019-2022. 19. Wecker, E., Schimpl, A. & Huinig, T. (1977) Nature (London)
269,598-600. 20. Hunsmann, G., Moennig, V. & Schafer, W. (1975) Virology 66,
327-329. 21. Friend, C. & Rossi, G. B. (1968) Int. J. Cancer 3,523-529.
