Proc. Nati. Acad. Sci. USAVol. 74, No. 4, pp. 1697-1701, April 1977Immunology

Immunoprevention of x-ray-induced leukemias in the C57BL mouse(endogenous type-C virus/antiviral immunity/tumor immunity)

ROBERT L. PETERS*, BERNARD SASS*, JOHN R. STEPHENSONt, ISMAIL K. AL-GHAZZOULI*, SHIGEO HINOt,ROBERT M. DONAHOE*, MEIR KENDET, STUART A. AARONSONt, AND GARY J. KELLOFFt* Microbiological Associates, Walkersville, Maryland 21793; and t Laboratory of RNA Tumor Viruses, National Cancer Institute, Bethesda, Maryland 20014

Communicated by Robert J. Huebner, January 18, 1977

ABSTRACT An attempt to prevent irradiation-inducedthymic lymphomas in C57BL mice was made by inducing activeimmunity to endogenous type-C virus with inactivated Rauschermurine leukemia virus (MuLV) or inactivated Gross MuLV orby transferring passive immunity to endogenous type-C viruswith goat anti-Gross-MuLV IgG. Control groups received thefollowing immunogen or treatment: inactivated simian sarcomavirus, complete Freund's adjuvant, normal goat IgG, and dilu-ent, in both irradiated and nonirradiated C57BL mice. Activeimmunity to the 70,000 molecular weight glycoprotein AKR-gp70 by immunization with Rauscher MuLV and passive im-munity to AKR-gp70 by passive transfer of goat anti-Gross-MuLV IgG was measurable throughout some of the latent pe-riod of tumor development; in these two groups a significantreduction in tumor incidence was observed, as compared to theother experimental and control groups. Thus, the present find-ings support the concept of a type-C virus etiology of irradia-tion-induced leukemias and demonstrate the applicability ofimmunologic techniques directed against the endogenoustype-C virus in the prevention of this disease.

The oncogenic potential of endogenous type-C viruses of themouse is implied by findings that naturally occurring type-Cviruses isolated from the animal (1-3) or activated from cellsin vitro (4) can induce lymphoreticular tumors in the same oran appropriately susceptible host strain. Recently, the researchof several laboratories has focused on immunoprevention oftumors thought to be mediated by endogenous viruses (5-7).Radiation-induced leukemia of the C57BL strain (8) providesan excellent model for such immunoprevention efforts. Kaplanand coworkers have presented a large body of experimentalevidence supporting the hypothesis that type-C virus, inducedby irradiation, is etiologically responsible for the subsequentdevelopment of thymic lymphoma (9). The present studies wereundertaken in an effort to develop immunologic approachesdirected against endogenous type-C virus that might lead toprevention of this and similar neoplasms.

MATERIALS AND METHODSMice. Strain C57BL/6N female mice, 30-36 days old, were

pooled and randomly distributed into experimental groups priorto treatment where they were individually identified andmonitored daily for illness or death.

Vaccines. Active series: Rauscher murine leukemia virus(R-MuLV) and Gross MuLV (G-MuLV) vaccines were pre-pared as previously described (6).

Passive series: Goat anti-G-MuLV serum was fractionatedand desalted as previously described (10). The resulting 560

Abbreviations: MuLV, murine leukemia virus; R-MuLV, Rauschermurine leukemia virus; G-MuLV, Gross murine leukemia virus; SSV,simian sarcoma virus; CFA, complete Freund's adjuvant; p12, currentnomenclature for the 12,000 molecular weight protein of MuLV; gp70,current nomenclature for the 70,000 molecular weight glycoproteinof MuLV; AKR-MuLV, naturally occurring murine leukemia virusof the AKR mouse.

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inactivated IgG preparation yielded a virus neutralizing end-point (10, 11) at a dilution of 1:800 and was not cytotoxic tonormal mouse cells in vitro when C57BL/6N mouse serum wasused as an endogenous source of complement.

Histopathology, Tissue Harvest, and Sera CollectionProcedures. A complete necropsy and sera collection wasperformed on each mouse at scheduled sacrifices or when themice were observed to be moribund or revealing signs of leu-kemia. Representative samples of all organs and tissues fromeach mouse were examined histopathologically. Additionally,lymphocyte preparations were prepared from about two-thirdsof each spleen, thymus, and bone marrow. Sera were diluted1:5 in phosphate-buffered saline and heat inactivated at 560for 30 min.

Virus Isolation Procedures. Lymphocyte suspensions frombone marrow, spleen, and thymus were inoculated at 5 X 106cells per 24 hr culture (Falcon, 60mm petri dish). For detectionof ecotropic MuLV, DEAE-dextran-treated SC-1 wild mouseembryo cells were used in the XC assay procedure (11, 12).Results were assessed after one passage at 7 days and inoculationof fresh 24 hr cultures. Presence of xenotropic MuLV was as-sessed by inoculation of lymphocyte suspensions (5 X 106 cells)from bone marrow, spleen, and thymus on a mink embryo cellline (13). Two subcultures were planted at 7 and 14 days, andat 21 days the culture supernatants were assayed for RNA-dependent DNA polymerase (reverse transcriptase) activity.Immunoassays. Radioimmunoassays for antibody to the

70,000 molecular weight glycoprotein gp7O from MuLV ofAKR mice (AKR-MuLV) and R-MuLV were described pre-viously (14) and were done at various scheduled intervals usinga 1:5 starting dilution of individual mouse sera. Assays forAKR-MuLV p12 antigen were done on cell suspensions con-taining 1 X 107 spleen, thymus, or marrow lymphocytes per mlusing previously described procedures (15). Selected sera, re-active in the radioimmunoassays, were tested for virus neu-tralizing ability by plaque reduction of MuLV in the XC test.A microadapted culture procedure for lymphocyte transfor-mation was used to detect cell-mediated responsiveness to virusvaccines and mitogenic stimuli as previously described (16).

Irradiationt. Mice received 150 rads (1.5 J/kg) of total-bodyx-irradiation in each of four weekly treatments as published byKaplan (17).

RESULTSExperimental Design. An outline of the experimental plan

that was followed to induce anti-MuLV immunity by activeimmunization with inactivated type-C virus or passive im-munization by treatment with anti-G-MuLV is shown in Table1. An effort was made to establish immune cells prior to the first

* Irradiation was generously done by Lieutenant Colonel Duane E.Hilmas, U.S. Army Medical Research Institute for Infectious Diseases,Fort Detrick, Frederick, MD.

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Table 1. Immunoprevention of x-ray-induced lymphoma-experimental design

X-ray (150 rads)tExperimental group numbers and immunogens Days administered* treatment days Test dayst

Active (x-ray) 1, R-MuLV; 2, G-MuLV; 3, SSV; 0, 6, 42, 56 14, 21, 28, 35 14, 35, 63, 97, 159, 2004, CFA

Active (control) 8, R-MuLV; 9, G-MuLV; 10, CFA Same as groups 1-4 None Same as groups 1-4Passive (x-ray) 5, Normal goat IgG; 6, goat anti- 13, 16, 20, 23, 27 14, 21, 28, 35 21, 42, 58, 104, 146

G-MuLV IgG; 7, diluent 30, 34, 37, 41, 48, 51Passive (control) 11, Anti-G-MuLV; 12, diluent Same as groups 5-7 None Same as groups 5-7

* All viral vaccines were given as 200 jg with complete Freund's adjuvant (CFA) on day 0, followed by 50 jg mixed with diluent on days 6, 42,and 56 (groups 1-3 and 8-9). CFA alone (groups 4 and 10) was given as 0.2 ml with 0.2 ml diluent. Groups 5-7 and 11-12 were given 0.2 ml oflisted passive treatment.

t 150 rads per dose.As described in Materials and Methods, animals were tested at the indicated days for infectious ecotropic and xenotropic type-C viruses; forMuLV gp7O antibodies; for MuLV-p12 antigens (days 58-63 only); for lymphoreticular and other neoplasms by complete histopathologicreview; and for immunologic injury and immune status by peripheral blood lymphocyte counts and lymphoblast transformation.

x-ray exposure by immunization with the inactivated viruspreparations on day 0 and day 6. No immunization attemptsby active means were made during the 4-week irradiationregimen (days 14-35), but booster immunizations were givenafter the irradiation regimen. The immunogens for the "active"immunization groups included inactivated R-MuLV and G-MuLV and control groups of inactivated simian sarcoma virus(SSV) and complete Freund's adjuvant alone (groups 14, Table1). Nonirradiated control groups receiving these immunogens(except SSV) were also included (groups 8-10, Table 1). In the"passive" immune groups, normal goat IgG, goat anti-G-MuLVIgG, and diluent (groups 5-7 and 11-12, Table 1) were given1 day before and 2 days after each of the four irradiationtreatments; and three additional injections were given, the lastone being 16 days after the last irradiation. Thus, during theirradiation regimen, a "passive" immunity treatment was givenevery 3-4 days. The experimental mice were sampled at ap-propriate time intervals for circulating antibody to MuLV,histopathologic changes, infectious and antigenic MuLV ex-

pression, and cell-mediated immune responses. Seventy-sixmice per group in the irradiated groups (groups 1-7) and 30mice per group in the nonirradiated groups (groups 8-12) were

studied. Sixteen mice per group were sacrificed for study byday 145, leaving 60 mice per group (in groups 1-7) at risk fortumor development.

Induction of Active Immunity to MuLV gp7O by Immu-nization with Inactivated Virus. In an attempt to induce activeimmunity to endogenous type-C virus, mice were given inac-tivated R-MuLV and G-MuLV. SSV and CFA alone were in-cluded as control immunogens (see Table 1, groups 1-4 and8-10). Immunizations were given at day 0 and day 6 and fol-lowed by the 4-week irradiation treatment (days 14-35).Booster immunizations were then given on day 42 and day 56.Test bleeds were obtained throughout the experiment and theability of the antisera to bind purified AKR-MuLV gp7O was

examined by radioimmunoassay (Table 2). Sera were tested atan initial dilution of 1:10 and serum titers presented are themeans of four mice per group at the respective days. The firstdetectable titers to AKR-MuLV gp7O were seen at day 35 in theR-MuLV- and G-MuLV-immunized groups, both x-irradiatedand nonirradiated. Serum endpoint titers in the R-MuLVgroups, both irradiated and nonirradiated, were significantlyhigher than the other groups, rising significantly until day 159to 1:600 and 1:1500, respectively. The serum titers to AKR gp7Oin the G-MuLV irradiated group was not observed to be sig-nificantly different from the SSV and CFA irradiated groups;however, all three groups gave reactivities that were higher than

sera from the diluent irradiated group. The nonirradiated R-MuLV and G-MuLV groups gave higher titers than their cor-responding irradiated groups; however, the converse was truefor the CFA groups. All virus-immunized groups showed adecline in serum titers at day 200 as compared to day 159 (Table2). Reactivities to homologous R-MuLV gp7O were measuredin the two R-MuLV-immunized groups giving serum endpointtiters 5- to 10-fold higher than to the heterologous AKR gp7Otiters. In selected serum samples from the R-MuLV groups thatwere reactive by radioimmunoassay, neutralizing activity wasdemonstrated against both live R-MuLV and AKR-MuLV atendpoint titers of 1:160 and 1:40, respectively, as measured byplaque reduction in the XC test.Measurement of AKR-MuLV gp7O Antibodies after Pas-

sive Immunization. Mean antibody titers (of four mice) toAKR-MuLV gp70 in mice receiving goat anti-G-MuLV IgGat 3- to 4-day intervals (see Table 1) are shown in Table 3. Titersof 1:60, 1:180, and 1:20 were detected at 1, 1, and 7 day intervalsafter IgG administration in the x-irradiated groups tested atdays 21, 42, and 58, respectively (Table 3). Comparable titers

Table 2. Circulating antibodies to AKR-MuLV gp7Oantigen in C57BL/6 mouse sera after activeimmunization with inactivated inocula

Mean antiserum titer for bindingAKR-MuLV gp70* on test day

Treat- X-ment ray 0 14 35 63 97 159 200

R-MuLV + <10 <10 40 90 400 600 120G-MuLV + <10 <10 20 40 80 80 40SSV + <10 <10 <10 20 80 80 40CFA + <10 <10 <10 <10 40 40 40Diluentt + <10 <10 <10 <10 <10 <10 NTR-MuLV - <10 <10 60 410 1200 1500 400G-MuLV - <10 <10 40 40 200 210 80CFA - <10 <10 <10 <10 20 20 20Diluentt - <10 <10 <10 <10 <10 <10 NT

NT, not tested.* Antisera titers above are based on four individual mice sampled ateach time period and are expressed as the reciprocal of the dilutionrequired to bind 10% of the 1251-labeled antigen.

tDiluent groups were bled and tested at test days designated in Table4-which were slightly different from designated test days listedin this table. These two diluent groups were listed also on this tablefor reference, even though sera were obtained on days 0, 21, 42, 58,104, and 146 as shown in Table 3.

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Table 3. Circulating antibodies to endogenous MuLVantigens after passive immunization with

normal G-MuLV immune goat sera

Mean antiserum titer for bindingAKR-MuLV gp7O* on test day

X-Treatment ray 0 21 42 58 104 146

Normal IgG + <10 <10 <10 <10 <10 <10Immune IgG + <10 60 180 20 <10 <10Diluent + <10 <10 <10 <10 <10 <10Immune IgG - <10 120 320 20 <10 <10Diluent - <10 <10 <10 <10 <10 <10

* Antisera titers above are based on four individual mice sampled ateach time period and are expressed as the reciprocal of the dilutionrequired to bind 10% of the l25I-labeled antigen.

were obtained in the pools of sera from the nonirradiatedcontrols receiving immune IgG. Because immune IgG wasadministered every 3-4 days from day 13 to day 51, it is as-sumed that circulating antiviral activity was continuouslymaintained from day 13 to day 58 (Table 3). No activity wasfound in the immune IgG group at days 104 or 146, nor was anyactivity detected in groups receiving normal goat IgG or dilu-ent, whether x-irradiated or not.Reduction of Irradiation-Induced Lymphomas and

Lymphocytic Leukemias in the Experimental Groups Hav-ing Actively or Passively Induced Antibody to AKR-gp7O. Asignificant reduction in total tumor incidence was observed inthe R-MuLV-immunized active group and the immune-IgG-treated passive group as compared to their respective controlsand the other experimental groups (Table 4). The final tumorincidences at day 270 revealed 16 tumors of 59 mice (27.1%)in the R-MuLV group and 16 tumors of 58 mice (27.6%) in theimmune IgG group as compared to 34 tumors of 56 mice(60.7%) in the diluent control group. By x2 analysis, these dif-ferences were significant at P = 0.002 and P = 0.001, respec-tively. Further, the tumor incidence of the R-MuLV group wassignificantly different from its adjuvant control group (CFA,group 4) at P < 0.05 and the immune IgG group was signifi-cantly different from its normal IgG control group (group 5)at the P < 0.05 level (Table 4).

Analysis of four of the experimental (control) groups (groups2-5 receiving G-MuLV + CFA, SSV + CFA, CFA alone, andnormal IgG) showed a striking similarity in their final percenttumor incidences which were, respectively, 48.3%, 47.8%,

48.1%, and 45.0%. These tumor incidences were consistently,though not significantly less than the diluent control group andwere statistically compared with the other appropriate exper-

imental and control groups as shown in Table 4.Examination of the latent period and time course of tumor

development in the irradiated experimental groups (Figs. 1 and2) showed that the tumrrors were first noted at about day 100, andin the diluent control group showed a steady rate of occurrencefrom days 130 to 230, at which time the rate of tumor occur-

rence dropped markedly. The two experimental groups inwhich significant reductions of final tumor incidences were

obtained showed a much slower rate of tumor occurrence

throughout and a "plateauing" of the tumor occurrence at days210-230. The four experimental groups having similar finaltumor incidences also showed similar latent periods and ratesof occurrence of tumor development.

Examination of Normal and Tumor-Bearing Animals forEndogenous Type-C Virus. Endogenous ecotropic type-C viruswas isolatable from the spleen, thymus, and bone marrow ofirradiated animals during the 4-week irradiation regimen andthroughout their latent period of tumor development. To ex-

amine the virus activation frequency in the diluent controlgroups, additional experiments using only this group were

conducted; and of a total of 51 normal animals examined fromdays 21 to 159, viruses were isolated from seven animals.Preparations from the spleens yielded the highest frequencyand all thymic isolates co-occurred with spleen isolates fromthe same animal. This low background incidence in the diluentcontrols did not allow definitive conclusions to be made aboutdifferences in virus incidence in normal animals among thedifferent experimental groups. Some interesting observations,however, were made; for example, the experimental groupsR-MuLV (group 1) and anti-G-MuLV immune IgG (group 6)yielded 0 isolates of 16 and 1 isolate of 16 animals, respectively,whereas age-matched diluent controls yielded 4 isolates of 16animals tested. Radioimmunoassays for ecotropic MuLV p12antigen were performed on extracts of spleen, thymus, and bonemarrow from all the experimental groups from days 21 to 159.The antigen titers of these tissues were found not to exceedbackground levels. Attempts to isolate xenotropic virus at thesetime periods from these three tissues yielded virus in only 4 of92 mice examined.

Animals sacrificed from days 161 to 270 allowed an exami-nation of endogenous virus incidence in tumor-bearing as wellas age-matched normal animals. The low incidence of virusisolatability from normal animals observed earlier in the ex-

periment continued throughout this later time interval and thus

Table 4. Effect of passive and active anti-MuLV immunization regimens on the incidence of leukemiain C57BL/6N mice after x-irradiation

No. with Probability of no difference versustGroup lymphoma/no. Treatment total at risk* % Diluent CFA Normal IgG

1 R-MuLV 16/59 27.1 P = 0.002t P < 0.05t2 G-MuLV 29/60 48.3 ns ns3 SSV 11/23 47.8 ns ns4 CFA 26/54 48.1 ns5 Normal IgG 27/60 45.0 ns6 Immune IgG 16/58 27.6 P = 0.001t P < 0.05t7 Diluent 34/56 60.7

* Number with lymphoma/total number at risk = number of mice with gross or histologically diagnosed lymphoma/number at risk after day 104.Day 104 was chosen because no lymphomas typical of x-ray induction were seen prior to this time in the animals sacrificed as scheduled, orin the animals under observation.

t Probability of no difference based on x2 analysis. ns = not significant at the P <0.05 level.

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FIG. 1. Cumulative incidence of lymphomas in irradiated C57BLmice after immunization with inactivated virus and control immu-nogens. R-MuLV, * *; G-MuLV, &---,&; SSV, 0 ---0; CFA, A-A;diluent, *-*.

no significant differences among the experimental groups couldbe established for these animals. A much higher incidence ofisolatable -virus was obtained,, however, from tumor-bearinganimals from the control groups, thus allowing the analysis ofdifferences in virus incidence and titer between tumor-bearinganimals from the control groups as compared to the immunizedgroups. Of considerable interest was the significant low inci-dence of isolatable virus from the tissues of animals in the ex-perimental groups (group 1, the R-MuLV immunized andgroup 6, the immune-IgG-treated) for which significant an-'tiviral antibody Lad been achieved at some time during thelatent period and where there had been a significant reductionof total tumor incidence. Table 5 shows this data with 0/15 and1/15 virus-positive tissues from tumor-bearing animals fromgroups receiving R-MuLV or immune IgG, respectively, versusI11/15, 7/18, and 8/20 virus-positive tissues from tumor-bearinganimals from groups receiving CFA, normal IgG, or diluent,respectively. The one isolate from group 6 (receiving immuneJgG) yielded 12 plaque-forming units, which was much lowerthan the titers generally obtained from isolates observed fromthe other groups.

DISCUSSIONThe data presented here establish that the incidence of irra-diation-induced thymic lymphomas and leukemias in this strainof mouse is significantly reduced in those mice having circu-lating antibody to endogenous ecotropic type-C virus duringtheir latent period of tumor development. This reduction oftumor incidence was observed whether circulating antibodywas induced by active immunization with inactivated type-Cvirus or was the result of passive treatment with anti-G-MuLVIgG. These data support the concept of the viral etiology ofthese irradiation-induced tumors and further establish thatneoplastic disease can be prevented by appropriate immuneregimens directed against the endogenous type-C virus (5-7).R-MuLV has been previously found to be a more effective

immunogen than G-MuLV in inducing neutralizing antibody

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15-

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90 110 130 150 170 190 210 230 250 270

Days Past Initial Immunization

FIG. 2. Cumulative incidence of lymphomas in irradiated C57BLmice after passive immune treatment with anti-G-MuLV IgG, normalgoat IgG, and diluent. Anti-G-MuLV IgG, 0-0; normal goat IgG,0-0; diluent, *-*.

to and resistance to challenge with a naturally occurringBALB/c leukemia virus (6). The greater immunogenicity ofR-MuLV is explained by its 100- to 200-fold greater gp7O ac-

tivity as determined by competition radioimmunoassay on

standardized virus preparations. These observations led to theinclusion of a R-MuLV immunogen group in these experimentseven though it was known that G-MuLV was more closely re-

lated serologically to Kaplan's original radiation leukemia virus.R-MuLV (given with CFA in the first injection) induced high

Table 5. Isolation of ecotropic MuLV fromlymphocytes of tumor-bearing and age-matched

irradiated non-tumor-bearing mice

Incidence of virus*

Age-matchedTumor- irradiatedbearing non-tumor-

Group mice bearing mice

ImmuneR-MuLV-(group 1) 0/15 0/14Anti-G-MuLV IgG(group 6) 1/15 0/9

ControlCFA alone (group 4) 11/15 3/13Normal goat IgG(group 5) 7/18 1/11

Diluent (group 7) 8/20 0/5

* Number of tissues positive for virus/number tested. Lymphocytesuspensions were prepared from the spleen, thymus, and bonemarrow tissues and 5 X 106 cells were inoculated onto duplicatecultures of 24-hr-old SC-1 cells. One plate was tested according tothe conventional XC procedure and the second plate was passagedat 7 days by scraping the cells into 0.6 ml of culture supernatant andbreaking up the cells using a 25 gauge needle and 1 ml syringe. Fresh,duplicate 24 hr plates of SC-1 cells were then inoculated with 0.2ml of the cell lysate and then tested 5 days later by the UV-XCassay.

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levels of anti-R-MuLV-gp7O and further was much more ef-fective in inducing antibody to AKR gp7O and in reducingtumor incidence than was G-MuLV. Hyperimmunization ofthe C57BL mouae with higher doses of G-MuLV does producea higher circulating anti-AKR-gp7O antibody (Kende et al.,unpublished data), as has also been observed in the NIH Swissmouse (18). Recent evidence exists (19) that the endogenoustype-C virus responsible for the radiation thymomas is not ofthe G-MuLV or AKR-MuLV envelope type. This suggests thatthe observed reduction in tumor incidence reported here is dueto a crossreactivity of the immunity induced by the effectiveregimens to the radiation leukemia virus of Kaplan.

Three of the four irradiated, active immunization groups-G-MuLV, SSV (both mixed with CFA with the first injection)and the CFA alone group (CFA given in the first injection,followed by diluent)-showed similar low levels of anti-AKR-gp7O activity, and they showed a striking similarity in their finaltumor incidence. The lower tumor incidence of these threegroups compared to the diluent group was apparent, althoughnot statistically significant and most probably occurred fromCFA activation of immune cells capable of responding to en-dogenous virus. Further, a fourth group showing a similar re-duction in tumor incidence was that which received normalgoat IgG, and when sera from these groups were retrospectivelyexamined for anti-AKR-gp7O activity of mouse gamma globulinorigin, they were found to have low level activity similar togroups 2-4. Of further interest was that although irradiationwas shown to activate low levels of isolatable endogenous type-Cvirus, it did not by itself result in humoral immunity to type-Cvirus by day 200 of the experiment (age of mice was 220-230days) even though it has been shown that this strain of mousedevelops naturally occurring humoral immunity to type-C viruslater in life (20, 21).

It should be noted that high antibody titers against AKR gp7Oin the R-MuLV immunized group were not obtained untilabout day 63 and were not sustained throughout the entire re-mainder of the latent period of tumor development. Similarly,during only a portion of the latent period (days 13 through 58)was a significant level of anti-AKR-gp7O activity maintainedin the only other experimental group (receiving anti-G-MuLVIgG) showing a significant tumor reduction. Thus, it might beassumed that, if this activity had been maintained (in a singleexperimental group) over a greater portion of the latent periodof tumor development, the tumor reduction would have beengreater than that observed.

Association of type-C viral antigens with thymomas by im-munoassay is hampered by the background activity of endog-enous xenotropic proteins of 30,000 and 12,000 molecularweight, p30 and p12 (22), and by the low percentage of virus-positive cells. These assays, therefore, can often be negative (23)on tissues that yield isolatable virus by infectious center assays.Infectivity assays yielded a low incidence of ecotropic type-Cviruses from normal tissues of irradiated control mice. Thisresulted in an inability to definitively establish reduction of thisactivity in immunized mice. The ecotropic virus activity in thetumor tissues was much higher, however, and made possiblethe observation that animals having antiviral immunity at sometime during their latent period show a very low incidence ofisolatable virus from their tumors relative to tumor-bearingcontrol animals. This suggests that either immunoselection

against or antigenic modulation of virus-positive tumor cellsoccurred in these animals (which is supported by recent datathat these mouse sera are cytotoxic Kende et al., unpublished).Such conclusions, however, are not possible until these or similartumors are also examined for the Kaplan radiation leukemiavirus (19), which now is known to have a different tropism andwas not detectable by these infectivity assays.The finding that tumors resulting from the cocarcinogenic

effect of a viral and physical agent can be prevented by im-munity directed against the endogenous type-C virus suggeststhe applicability of this approach in attempting the immuno-prevention of "naturally" occurring and "chemically" inducedtumors (24) known to express endogenous type-C virus infor-mation.

This work was supported by U.S. Public Health Service ContractN01-CP-33248 from the Division of Cancer Cause and Preventionwithin the Virus Cancer Program of the National Cancer Institute.

1. Gross, L. (1951) Proc. Soc. Exp. Biol. Med. 78,342-348.2. Lieberman, M. L. & Kaplan, H. S. (1959) Science 130, 387-

388.3. Peters, R. L., Spahn, G. J., Rabstein, L. S., Kelloff, G. J. &

Huebner, R. J. (1973) J. Natl. Cancer Inst. 51, 621-630.4. Stephenson, J. R., Greenberger, J. S. & Aaronson, S. A. (1974) J.

Virol. 13,237-240.5. Huebner, R. J., Gilden, R. V., Lane, W. T., Toni, R., Trimmer,

R. W. & Hill, P. R. (1976) Proc. Natl. Acad. Sci. USA 73,620-624.

6. Kelloff, G. J., Peters, R. L., Donahoe, R. M., Ghazzouli, I., Sass,B., Nims, R. M. & Huebner, R. J. (1976) Cancer Res. 36, 622-630.

7. Huebner, R. J., Gilden, R. V., Toni, R., Hill, R. W., Trimmer, R.W., Fish, D. C. & Sass, B. (1976) Proc. Natl. Acad. Sci. USA 73,4633-4635.

8. Kaplan, H. S. (1948) J. Nati. Cancer Inst. 9,55-56.9. Kaplan, H. S. (1967) Cancer Res. 27, 1325-1340.

10. Hebert, G. A., Pelham, P. L. & Pittman, B. (1973) Appl. Micro-biol. 25,26-36.

11. Rowe, W. P., Pugh, W. E. & Hartley, J. W. (1970) Virology 42,1136-1139.

12. Klement, V., Rowe, W. P., Hartley, J. W. & Pugh, W. E. (1969)Proc. Natl. Acad. Sci. USA 63, 753-758.

13. Peebles, P. T. (1975) Virology 67,288-291.14. Hino, S., Stephenson, J. R. & Aaronson, S. A. (1976) J. Virol. 18,

933-941.15. Tronick, S. R., Stephenson, J. R. & Aaronson, S. A. (1973) Virology

54, 199-206.16. Donahoe, R. M., Peters, R. L., VanVleck, R., Huebner, R. J. &

Kelloff, G. J. (1976) J. Natl. Cancer Inst. 56,51-57.17. Kaplan, H. S. & Brown, M. B. (1952) J. Natl. Cancer Inst. 13,

185-208.18. Lee, J. C., Ihle, J. N. & Huebner, R. (1977) Proc. Natl. Acad. Sci.

USA 74,343-347.19. Decleve, A., Lieberman, M., IhIe, J. N. & Kaplan, H. S. (1976)

Proc. Natl. Acad. Sci. USA 73, 4675-4679.20. Ihle, J. N., Domotor, J. J., Jr. & Bengali, K. M. (1976)J. Virol. 18,

124-131.21. Stephenson, J. R., Peters, R. L., Hino, S., Donahoe, R. M., Long,

L. K., Aaronson, S. A. & Kelloff, G. J. (1976) J. Virol. 19,890-898.

22. Kelloff, G. J., Peters, R. L., Donahoe, R. M., Stephenson, J. R. &Aaronson, S. A. (1976) J. Nati. Cancer Inst. 57,85-89.

23. Ihle, J. N., Joseph, D. R. & Pazmino, N. H. (1976) J. Exp. Med.144, 1406-1423.

24. Whitmire, C. E. & Huebner, R. J. (1972) Science 177,60-61.

Immunology: Peters et al.

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Proc. Natl. Acad. Sci. USA 76 (1979) 4157

Correction. In the article "Cellular energy metabolism,trans-plasma and trans-mitochondrial membrane potentials,and pH gradients in mouse neuroblastoma" by C. Deutsch, M.Erecin'ska, R. Werrlein, and I. A. Silver, which appeared in theMay 1979 issue of Proc. Natl. Acad. Sci. USA (76, 2175-2179),the authors request that the following changes be noted. Onpage 2177 in lines 8 and 9 of the left-hand column, the mem-brane potentials should be "-29.6 i 3.0 mV (n = 6) (SCN)" and"-77.0 ± 1.5 mV (n = 8) (TPMP)."

Correction. In the article "Immunoprevention of x-ray-inducedleukemias in the C57BL mouse" by Robert L. Peters, BernardSass, John R. Stephenson, Ismail K. Al-Ghazzouli, Shigeo Hino,Robert M. Donahoe, Meir Kende, Stuart A. Aaronson, and GaryJ. Kelloff, which appeared in the April 1977 issue of Proc. Natl.Acad. Sci. USA (74, 1697-1701), the authors request that thefollowing correction be noted. On page 1697, line eight of theleft hand column, an additional reference should be cited:Ferrer, J. F., Lieberman, M. & Kaplan, H. S. (1973) Cancer Res.33, 1339-1343.

Correction. In the article "Evolutionary change in 5S RNAsecondary structure and a phylogenic tree of 54 5S RNAspecies" by Hiroshi Hori and Syozo Osawa, which appeared inthe January 1979 issue of Proc. Natl. Acad. Sci. USA (76,381-385), the authors request the following change. On page382, in line 1 of the legend for Fig. 1, "(A) E. coli prokaryotic116-N type; (B) B. subtilis prokaryotic 120-N type" should read"(A) B. subtilis prokaryotic 116-N type; (B) E. coli prokaryotic120-N type."

Corrections