Expression of natural antibodies against endogenous and horizontally transmitted macaque retroviruses in captive primates

‘IROLOGY

112, 49-61

(1981)

Expression of Natural Antibodies against Transmitted Macaque Retroviruses DONALD Biological

Carcinogenesis

Program,

L. FINE’

LARRY

AND

NCI Frederick

Endogenous in Captive

Cancer

Accepted December

and Horizontally Primates’

0. ARTHUR

Research Center, Frederick,

Maryland

21701

19, 1980

To determine the prevalence of retrovirus antibodies in captive primate populations and to identify factors that influence antibody responses, we analyzed sera from seven geographically separated primate colonies for antibodies to the genetically [baboon endogenous virus (BaEV), squirrel monkey retrovirus (SMRV), and macaque endogenous viruses (MMC-1 and MAC-l)] and horizontally [Mason Pfizer monkey virus (MPMV) and gibbon ape lymphoma virus (GaLV)] transmitted primate retroviruses. Antibodies were detected with a solid-phase radioimmunoassay in which iodinated staphylococcal protein A is used for precipitation of immunoglobulins. Naturally occurring antibodies to MPMV were found in sera of Macaca mulatta from seven geographically separated primate colonies and in the sera of three different macaque species. The predominant reactivity of these natural sera was directed against antigens shared by MPMV, BaEV, and SMRV. Several of these sera were found to have MPMV-neutralizing activity. Antibodies to the endogenous Macaca mulatta virus, MMC-1, were found in three species of macaques and in Macaca mulatta sera from five of the seven colonies tested. These positive sera were directed predominantly against a 69,000 molecular weight protein and against determinants shared by both MMC-1 and MAC-l, an endogenous virus of Macaca arctoides. Antibody expression to MMC-1 appears to be both sex and age related, whereas antibody expression to MPMV in the same monkey population was not.

D infectious retroviruses are horizontally transmitted from animal to animal under natural conditions. Although nucleic acid sequences related to GaLV are not detected in the DNA of primate cells (Scolnick et al., 1974; Benveniste et al., 1974a; Wong-Staal et al., 1975), nucleic acid sequences partially related to MPMV are present in the DNA of all Old World monkeys (Drohan et al., 1977; Benveniste and Todaro, 1977). Based on the latter findings, it was suggested that MPMV was derived from an endogenous virus of Old World monkeys, in particular the langur monkey (Benveniste and Todaro, 1977). The endogenous or genetically transmitted retroviruses, which comprise the second major class of primate retroviruses, are represented as multiple-copied viral genes in the cellular DNA of their respective host species. Five distinct groups have been identified: (i) type D virus (POl-Lu) isolated from the langur monkey

INTRODUCTION

Reverse transcriptase-containing viruses (retroviruses) have been isolated from a variety of avian and mammalian species. In primates, two major classes of retroviruses have been described: infectious and endogenous. The infectious retroviruses from Old World and New World monkeys and apes include the type C viruses isolated from gibbon apes (GaLV) (Kawakami et al., 1972) and a woolly monkey (SSV-1) (Theilen et al., 1971) and the type D Mason-Pfizer monkey virus (MPMV) isolated from rhesus monkeys (Jensen et al., 1970). Both type C and type i The U. S. Government’s right to retain a nonex:lusive royalty-free license in and to the copyright :overing this paper, for governmental purposes, is acknowledged. ’ Author to whom requests for reprints should be addressed. 49

0042-6822/81/090049-13$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

50

FINE

AND

(Benveniste and Todaro, 1977, Todaro et al., 1978a), which is closely related to MPMV as determined by immunological and structural analyses (Bryant et al., 1978), (ii) type D virus (SMRV) isolated from squirrel monkeys (Heberling et al., 19’77; Schochetman et al., 1977; Schochetman and Fine, 1978; Todaro et al., 1978a), (iii) type C virus (OMC-1) isolated from an owl monkey (Todaro et al., 1978c), (iv) type C virus (BaEV) isolated from baboons and related species (Benveniste et al., 1974b; Todaro et al., 1974; Todaro et al., 1976), and (v) type C virus (MMC-1, MAC1) recently isolated from the Asian macaque species (Todaro et al., 1978b; Rabin et al., 1979). Although MMC-1 and MAC-l are indistinguishable, both antigenically and based on nucleic acid sequence homology (Rabin et al., 1979), the RNA genomes of the other groups of endogenous primate type C and type D retroviruses are distinguishable from one another as determined by nucleic acid hybridization criteria (Benveniste and Todaro, 1973, 1976, 1977; Bryant et al., 1978). Type C and D viral structural proteins are unique as demonstrated by both biochemical and immunological analyses. Consequently, members of each group of endogenous primate retroviruses do not cross-react in species-specific radioimmunoassays (RIAs) (Tronick et al., 1974; Schochetman et al., 1977; Stephenson et al., 1976; Fine and Schochetman, 1978). However, recent studies have shown that the envelope glycoproteins of MPMV, PO-lLu, SMRV, and BaEV share antigenic determinants that are not detectable in the envelope glycoproteins of MAC-l and OMC-1 (Bryant et al., 1978). Serological assays permitting detection of retrovirus structural proteins have been used to survey for virus gene expression in various animal systems. Naturally occurring antibodies to the horizontally transmitted viruses, MPMV and GaLV, have been demonstrated respectively in macaques and gibbon apes (Fine et al., 1978b; Kawakami et al., 1972). Similarly, antibodies to the endogenous virus, SMRV, in its host species have been reported (Devare et al., 1978a). In this report, we

ARTHUR

describe the natural occurrence of precip itating antibodies that recognize envelop glycoproteins of the endogenous macaqu virus MMC-1. Additionally, to gain a bet ter understanding of the prevalence of ret rovirus expression in primates, we hav analyzed sera from seven geographicall: separated primate colonies for antibodie to the horizontally transmitted viruse (MPMV and GaLV), as well as to the ge netically transmitted retroviruses (BaEV SMRV, MMC-1, and MAC-l). Five specie of nonhuman primates were included il the analysis. In macaques, in which w examined prevalence of antibodies to botl horizontally transmitted (MPMV) and en dogenous retroviruses (MMC-l), variou physiological and environmental factor including species, sex, age, caging, am breeding status were evaluated with re gard to their influence on antibody expres sion. MATERIALS

AND

METHODS

Sera. Primate sera were generously pro vided by the following primate center (responsible investigator): Litton Bionet its, Kensington, Maryland. (Dr. J. Cic manec); California Primate Center, Davis California (Dr. M. Nold); Delta Regiona Primate Center, Covington, Louisiana (Dr P. Gerone); Hazelton Primate Colony Springfield, Virginia (Dr. J. Dalgard) Rockefeller University, New York, Nev York (Dr. K. Sundaram); University o Cincinnati Childrens Hospital Researcl Foundation, Cincinnati, Ohio (Dr. R. Frad kin); and Yerkes Primate Center, Atlanta Georgia (Dr. H. McClure). All sera wer shipped frozen to the Frederick Came, Research Center. Test viruses. Purified retroviruses were provided by the Viral Resources Section Frederick Cancer Research Center. Thes viruses, produced from their respective host cells, included GaLV from 6-G-l (gib bon ape lymphoblast) cells, MMC-1 fron CFK2th (canine thymus) cells, MAC-: from CFK2th and A549 (human alveolar carcinoma) cells, BaEV and MPMV fron A204 (human rhabdomyosarcoma) cells

RETROVIRUS

ANTIBODIES

SMRV from D1’7 (canine osteosarcoma) cells, mouse mammary tumor virus (MMTV) from MmEimt/q (C3H mouse mammary tumor) cells, and Rauscher murine leukemia virus (R-MuLV) from JLSV9 (BALB/c mouse bone marrow) cells. For iodinated protein A (IPA) assays, concentrates (1000X) of these viruses suspended in TNE buffer (0.01 M Tris-HCl, pH 7.4, 0.01 M NaCl, and 0.001 M EDTA) were disrupted by three cycles of freezethawing with intervening sonications (30 set at 30 mA). Disrupted virus was diluted in phosphate-buffered saline to a final concentration of 400 pg/ml. Radioimmune precipitation (RIP) assay. Precipitating antibody to MPMV was measured by intact virus particle RIP assays. Intact MPMV was radiolabeled with “‘1 using lactoperoxidase (Cardiff et al., 1974). The labeled virus was dialyzed extensively against RIP buffer (0.01 1M TrisHCl, pH 7.4, 0.01 M NaCl, and 0.001 M EDTA) and then banded on a 15-50% (w/ w) sucrose gradient; the radioactive peak at 1.17 g/ml was collected. Specific activity of these virus preparations averaged 1 x 105 cpm/pg of protein. The labeled virus was 97% precipitable with 10% TCA, and completely precipitable by excess hyperimmune rabbit antibody to sucrose-banded virus. The RIP assay consisted of incubating 100 ~1 of ‘251-MPMV (15,000-20,000 cpm) with 100 ~1 of diluted monkey serum for 1 hr at 37”. A volume of 300 ~1 of goat anti-rhesus monkey IgG (diluted 1:25 in RIP buffer) was added, and incubation was continued at 37” for 1 hr and overnight at 4”. After incubation, 1 ml of RIP buffer was added, and the immune precipitates were collected by centrifugation at 2500 g for 30 min. Supernatants were aspirated, and the radioactivity in the immune pellets was determined. 1251-Labeled protein A (IPA) assay for antiviral antibodies. Antiviral antibodies were detected by a binding assay with 1251labeled protein A (IPA) from Staphylococcus aureu.s (Nowinski et al., 1979). Viruses were adjusted to a concentration of 10 pg/ml. Briefly, 0.5 pg of virus in 50 ~1 of phosphate-buffered saline (PBS, pH 7.2) was absorbed to the individual wells of a

IN PRIMATES

51

microtiter plate by incubation overnight at 37”. The following morning, the wells of the plate were blocked from further nonspecific protein adsorption by a 2-hr incubation with 125 ~1 of 5% bovine serum albumin (BSA) in PBS, pH 7.2. The IPA assay usually was performed in two steps: (i) 50 ~1 of fluid containing antibody was incubated in each of the virus-adsorbed wells for 45 min at 37”. The wells were washed three times with PBS containing 1% BSA to remove all nonbound immunoglobulins. (ii) IPA (lo5 cpm) in 50 ~1 PBS was added to each of the virus-adsorbed wells for 45 min at 37”. The residual nonbound IPA was then removed from the wells by a wash with PBS, and the immune reactions were detected by 24 to 48 hr autoradiography of the IPA-treated microtiter plates on Kodak NS-2T film. All virus antigens were reactive as demonstrated by the use of hyperimmune sera. Because IPA binds preferentially to certain immunoglobulin classes, a secondary system was employed to detect non-IPAbinding immunoglobulins. For this, an intermediate incubation was performed with a goat antiserum that was prepared against rhesus monkey IgG. This intermediate incubation (between steps 1 and 2) was performed for 1 hr at 37” with 50 ~1 of l/1000 diluted rabbit antiserum; nonbound rabbit antibodies were removed from the wells by a wash with PBS containing 1% BSA, and the assay was continued by the addition of IPA as described above. Polyacrylamide gel electrophoresis (PAGE). Electrophoretic separation of polypeptides by sodium dodecyl sulfate (SDS-PAGE) was based on the methods of Maize1 (1969) and previously detailed (Arthur et al., 1978). Localization of radiolabeled proteins was accomplished by autoradiography as described by Arthur and Fine (1979). Protein content was determined according to Lowry et al. (1951) using BSA as a standard. Iodination of MMC-1 virus. MMC-1 lysed with 1% Triton X-100, 0.5% sodium desoxycholate, and 3 M urea was radiolabeled with 1251using chloramine-T, and immune precipitation of lysed 1251-MMC-1 by hy-

52

FINE

AND

perimmune rabbit anti-MMC-1 sera of Macaca sp. sera was performed according to the procedure originally described for MMTV (Arthur and Fine, 1979). Immunoprecipitated proteins were resolved by SDS-PAGE, and radiolabeled proteins were localized by autoradiography. Data analysis. Results on antibody frequencies in various primate species were analyzed by the method of contingency table analysis using the Fisher exact test in lieu of x2 (Fleiss, 1973). Trend test probabilities were prepared using the program of Thomas et al. (1977). RESULTS

Detection of Antibodies to Retroviruses Subhuman Primate Sera

in

In previous studies, antibodies precipitating type D retroviruses were detected in sera of both MPMV-inoculated and noninoculated primates using radiolabeled purified virus proteins and intact virus preparations (Fine et al., 1978b, c; Devare et al., 19’78a). Because this sensitive RIP assay is not readily applicable for screening large numbers of sera for antibodies against a variety of retroviruses, we adapted a solid-phase immunoassay that utilizes iodinated staphylococcal protein A (IPA) for detection of antibodies to retroviruses in subhuman primate sera. Sera from MPMV-inoculated and noninoculated macaques were tested for serological reactivity in the IPA against GaLV, MMC1, BaEV, MPMV, SMRV, and MMTV. Examples of IPA reactivities are shown in Figs. 1 and 2. The anti-primate retrovirus activity of a small number of sera (25 of 660) which could be removed by absorption with murine retroviruses, fetal calf serum, or uninfected cells were apparently directed against heterophile antigens and were scored negative in the study. Sera from both MPMV-inoculated and noninoculated rhesus monkeys (Macaca muZatta) and noninoculated bonnet monkeys (Macaca radiata) were found to have antibodies to MPMV (Table 1). Sixteen percent of these sera exhibited cross-reactivity with BaEV and 12% were positive with

ARTHUR

84719

B4720

B4722

84993

85069

FIG. 1. Autoradiogram of IPA showing reactivity of sera from MPMV-inoculated Mucaca mulatta with retrovirus antigens. B4719, B4720, B4722-sera collected 220 weeks postinoculation with MPMV; B5069serum collected 97 weeks postinoculation with MPMVtransformed rhesus foreskin cells; B4993-serum collected from control monkey 16 weeks after inoculation with rhesus meningioma tissue.

SMRV. This type of reactivity was found both in colony-born and imported animals housed in two geographically separated primate colonies. Of 51 Macaca mulatta inoculated with MPMV, 29 (56%) were positive for MPMV antibody. Stress of inoculation did not appear to induce expression of MPMV cross-reactive antigens since only 2 of a group of 19 (10%) mislymcellaneous-inoculated (Burkitt’s phoma, Herpes simplex types I and II, and rhesus meningioma tissue) Macaca mulatta had sera which reacted with MPMV. Reactivities to MMC-1 were also detected in Macaca mulatta, Macaca radiata, and Macaca arctoides. The reproducibility of the serological reactivity was demonstrated in sequential sera samples collected from several macaques over a 15month period (i.e., Mmu 8016) (Table 1). An example of the titer and absorption specificity of one of the MMC-1 positive sera is shown in Fig. 3. Demonstration of Shared Antigenic Determinants of Primate Retroviruses We previously found that BaEV, SMRV, MPMV, and the endogenous cat virus,

RETROVIRUS GalV

MMC-I

BaEV

ANTIBODIES

MPMV SMRV

Mmu 264 M!TW 566 Mmu 693 Mmu 960

1246 Mmu 1927 Mill&l 2722 Mmu 2912

FIG. 2. Autoradiogram of IPA of Macaca mulatta serum with test retrovirus antigens. Sera were collected from singly caged noninoculated monkeys at the California Primate Center. Mmu 586 (165-monthold) and Mmu 893 (156-month-old) were colony born; other animals in test were imported.

RD114, share common determinants on their gp70 molecule for binding to cell surface receptors (Fine et al., 1980). With the IPA we found that macaque sera similarly recognize the same three primate viruses (Table 2) and RD114 (data not shown). Animal descriptions and antigens used for absorptions are indicated with the results. Several of the sera from both MPMV-inoculated and noninoculated Macaca muZatta (B1596 and B6931) that showed reactivity for BaEV, MPMV, and SMRV failed to react after absorption with MPMV. Recognition of common determinants shared by these three viruses was also observed for sera from Macaca radiata (Mra16149). Reciprocal absorptions of this serum by MPMV, SMRV, or BaEV removed reactivity to all three viruses, whereas absorption with MMTV (Fig. 4) or MMC-1 (data not shown) did not. Sera from both ikfacaca mulatta (Mmu7492 and Mmu8008) and Macaca arctoides (Msp235) that reacted only with MMC-1 were rendered nonreactive by absorption with MMC-1. These sera appear to recognize determinants common to both MMC-1 and MAC-l because absorption with either virus removed reac-

IN PRIMATES

53

tivity to MMC-1. Common determinants were virus specific rather than cell specific, as absorptions with either canine- or human cell-produced MAC-l virus did not alter the unabsorbed or absorbed serological reactivities (Fig. 3). Additionally, absorption of these sera with SMRV, MuLV, and MPMV (data not shown) did not remove the serum reactivity with MMC-1 (Table 2). Only 1 positive serum out of 11 tested reacted with MAC-l grown in dog cells but not with MAC-l grown in human cells. We speculate that these sera recognize cellular-specific post-translational modifications (in carbohydrate addition) similar to the nonspecific reactivity of human sera to retrovirus (Snyder and Fleissner, 1980; Barbacid et al., 1980). Any sera showing this reactivity were considered negative. Characterization of MMC-1 Antigens Reactive with Natural Antibody in Macaque Sera We have found that naturally occurring precipitating antibody to MPMV in Macaca mulatta sera recognized the envelope glycoprotein gp70 (unpublished data). In characterizing the specificity of natural antibody to MMC-1 virus, we determined which virion proteins of MMC-1 were precipitated by sera from noninoculated macaques. Sucrose gradient-purified MMC-1 virus was disrupted and iodinated and then the radiolabeled proteins were precipitated by sera from several macaque species, as well as by hyperimmune rabbit serum. Radiolabeled proteins precipitated by the sera were separated by SDS-PAGE and identified by autoradiography (Fig. 5). Rabbit anti-MMC-1 serum precipitated all of the major MMC-1 proteins, whereas IPA-positive sera from Macaca arctoides 242 and Macaca mulatta 8016 precipitated predominantly the 73,000 molecular weight protein. The latter sera also precipitated a smaller protein of approximately 18,500 molecular weight. Two sera (Macaca radiata 16149 and Macaca mulatta 7261) which did not react with MMC-1 in the IPA assay did not precipitate any radiolabeled proteins. One of these two sera

M. mulatta

M. mulatta

M. mulatta

893

8016

16149

16137

16150

242

B5074

142A

MMU

MMU

MRA

MRA

MRA

MSP

radiata

radiata

CPCd

CPCd

CPCd

CPCd

CPC

CPC”

LBI”

LBI”

LBIC

LBI”

Primate colony”

OF MACAQUE

born/

Primate Center. of titer which

Imported, female, noninoculated

Imported, female, noninoculated

Imported, female, noninoculated

Imported, female, noninoculated

Imported, female, noninoculated

represents

nonbreeder/

breeder/

breeder/

breeder/

breeder/

born,

female/noninoculated

156-week-old, colony female nonbreeder/ noninoculated

Imported,

Virgin female, colony born/ inoculated at birth with MPMV-infected cells

Virgin female, colony MPMV inoculated

born/

description/treatment

TO RETROVIRUS

Virgin female, colony MPMV inoculated

Animal

SERA REACTIVITIES

a LBI, Litton Bionetics; CPC, California *Numbers in parentheses are reciprocals +l to +4 with +4 being greatest intensity. ’ Indicates animal singly caged. d Indicates animal group caged.

M. arctoides

M. radiata

M

M

M. mulatta

M. mulatta

B4720

Species

M. mulatta

No.

B4719

Animal

SUMMARY

highest

1

-

+4 (400) +3

-

-

Quarantine 30’8

3/78 6/79

serum

3/78

6/79

3/78

3/78

dilution

+2 (200) -

+4 (200)

-

reaction;

-

positive

+2

-

-

1 week postinoculation; 39 weeks postinoculation

giving

-

-

-

220 weeks postinoculation

-

intensity

to*

-

+2

+1

reactions

+2 (200)

-

+3 (50)

+1

+2

-

SMRV

ASSAYS

of positive

-

-

-

+2 (400)

+I

+2

+3

+1 (100)

+3

+4

f2

MPMV

reactivity

A (IPA)

+2

+1

+2

-

-

+3

-

+2

-

-

220 weeks postinoculation

-

IPA

PROTEIN

BaEV

STAPHYLOCOCCAL

MMC-1

collection

IN IODINATED

GALV

Serum

ANTIGENS

TABLE

scored

-

-

-

-

-

-

-

-

MMTV

ia 2 s

z 3

RETROVIRUS Mmu

ANTIBODIES

7492

FIG. nation

3. Autoradiogram of IPA showing dletermiof endpoint serum-binding titer for Macaca mulatta 7492 with MMC-1 virus. Unabsorbed serum is reactive with both MMC-1 and MAC-l but not RMuLV. Absorption of serum with MMC-1 remloves all reactivity for MMC-1 and MAC-l. (Test antigens show MAC-l grown in either human or dog cells.)

(Mra 16149) which did not recognize MMC1 was reactive in the IPA with BaEV, MPMV, and SMRV (Tables 1 and 2). Distribution of Antibodies to MMC.-1 and MPMV in Sera of Captive Primate Populations Sera from 507 animals were tested for antibody to MMC-1 and MPMV (Table 3). These sera represented seven primate colonies from various geographic locations. The monkeys were representative of four macaque species and one species of Cercopithecus. These animals were primarily from breeding (Litton Bionetics, Hazelton Primate Colony), endocrinological (Rockefeller University, University of Cincinnati), and long-term holding studies (Yerkes Primate Center). None of the animals had been inoculated against retrovirus. MPMV antibody-positive animals were found in all seven colonies. Of the 56’7 ani-

IN PRIMATES

55

mals tested, 82 (17%) were positive for MPMV. The California Primate Center had the highest percentage of MPMV-positive animals of the four species of macaques tested. Macaca radiata was the most positive (43%) macaque species, whereas Macaca arctoides was the least (no antibodies were found in 18 animals tested). In addition, the geometric mean antibody titer to MPMV of the aged-matched animals was higher in Macaca radiata (1:328) than in Macoxa mulatta (1:50). Antibodies to MMC-1 virus were found in five of the seven colonies tested. Of the total of the 507 sera tested, 45 were positive to MMC-1 virus (9%). Antibodies to MMC-1 virus were found in three (Macaca mulatta, Macaca radiata, Macaca arctoides) of the four macaque species tested. Of these, Macaca arctoides had the highest percentage (17%) positive sera. The highest frequency of MMC-1 sera reactive with MMC-1 virus was from a group of recently imported Cercopithecus aethiops at the Hazelton Primate Colony. No MMC-1 positive sera were found in a group of New World monkeys (two Aotus trivirgatus, six Ateles sp., and four Saimiri sciureus) housed at the same colony. No MMC-lpositive sera were found in animals from the University of Cincinnati, but this might be expected because only seven animals were tested from this colony. No antibodies to MMC-1 were detected in 24 Macaca mulatta at the Delta Primate Center, which were a group of young, recently imported animals. In Macaca mulatta the frequency of antibody-positive sera to either MPMV or MMC-1 was not significantly affected by whether the animal was imported or colony born (e.g., at Litton Bionetics, Inc.). Similar results were found for animals at the California Primate Center. Of 38 animals having MPMV antibodies, 26% were colony born and 28% were imported. Of the 16 animals having MMC-1 antibodies, 11% were colony born and 17% were imported. No antibodies to either MMC-1 or MPMV were detected in five lymphoma-bearing Macaca mulatta from the California Primate Center. Incidences of antibody in two

56

FINE

AND

ARTHUR

TABLE IPA

SPECIFICITY

ESTABLISHED

BY SERUM

2

ABSORPTION

Species

Bl596

B6931

MSP235

MRA16149

M. mulatta

M

mulatta

M. arctoides

M. radiata

Description Burkitt’s lymphoma MPMV-inoculated (colony born)

COIOW

and female

Normal male breeder (colony born)

Normal female breeder (imported)

Normal female breeder (imported)

LBI/MRI”

LB1

CPCb

CPC

LrAnimal born at Litton Bionetics, Inc. (LBI), Kensington, Md., inoculated Institute (MRI) at 6 years of age, then inoculated with MPMV. b Indicates California Primate Center, Davis, Calif.

groups of irradiated Macaca mulatta, held at the Yerkes Primate Center and Litton Bionetics, Inc. Primate Colony, were comparable to those observed in normal animals in the same colony. Based on these data, it would appear that irradiation of these animals did not result in increased retrovirus expression or increased susceptibility (antibody development) to retrovirus expression. Additional environmental and physiological factors that could influence expression of retrovirus proteins and, consequently, antibodies to these proteins were examined in a group of 171 macaques housed at the California Primate Center. There were no significant differences in antibody frequencies to each of the primate retroviruses (GaLV, MMC-1, BaEV, MPMV, and SMRV) in animals caged singly as compared to animals caged in groups. Comparison of age-matched male and female macaques revealed that females have a higher frequency of antibody response (P = 0.052) to MMC-1, whereas frequency of antibody expression to the other retroviruses was the same for both males and females. Analysis of the breed-

VARIOUS

Serum unabsorbed/ absorbed with

Animal NO.

WITH

RETROVIRUSES IPA reactivity

to

MMC-1

BaEV

MPMV

SMRV

Unabsorbed MPMV MMC-1 MMTV

-

+ + +

+ + +

+ + +

Unabsorbed MPMV MMC-1 MMTV

-

+ +

+ + +

+

Unabsorbed MMC-1 MAC-l SMRV R-MuLV

+ -

-

-

+ +

-

Unabsorbed MPMV SMRV BaEV MMTV

-

+

at LB1 with Burkitt’s

-

+ -

+ -

+

Iymphoma,

+ +

transferred

+

+

to Mason Research

ing status of female Macaca mulatta (including pregnant animals) revealed no significant differences between breeders and nonbreeders in antibody frequency to the retroviruses tested. Macaca mulatta of various ages (16 to 144 months old) were also tested. Although frequency of antibody to MMC-1 showed a significant in-

FIG. 4. Demonstration of specificity of broadly reactive Mucaca radiata (Mra 16149) serum by absorption with MPMV, BaEV, and SMRV. Absorption of serum with MMTV did not remove cross-reactivity.

RETROVIRUS

ANTIBODIES

IN

PRIMATES

57

73,000 71,000

-26,500 .22,000 '18,500 -12,500 -11,000

FIG. 5. Autoradiogram of SDS-PAGE resolution of immunoprecipitated ‘251-MMC-1 proteins. End lanes contain lysed ‘251-MMC-1 virus. RolMMC-1, hyperimmune rabbit anti-MMC-1 serum; Mmu 8016, M. mulatta and Msp 242, M. arctoides (MMC-l-positive serum); Mra 16149, M radiata (MPMV-positive serum); Mmu 7261, M. mulatta (negative serum). Approximate molecular weights shown in right column.

crease with age (test for positive trend; P = 0.049), frequency of antibodies to other retroviruses was not age related.

Neutralizing

Antibody

to MPMV

MPMV infection of KC cells results in production of multinucleated syncytia (Ahmed et al., 1974; Rand et al., 1974). This biological assay provides a means for detecting MPMV-neutralizing antibody (Fine et al., 1980). Sera from both normal and MPMV-inoculated monkeys were tested to determine if macaques could elicit neutralizing antibodies to MPMV. As shown in Table 4, MPMV inoculation of Macaca mulatta induces neutralizing antibodies to MPMV corresponding to high-titered precipitating antibodies detected by both RIP and IPA assays. Neutralizing antibodies were also detected in several uninoculated monkeys although titers were not as high as in MPMV-inoculated monkeys. Correspondingly lower RIP and IPA titers were observed for the same animals. DISCUSSION

Our previous reports showed the occurrence of natural antibodies to the Mason-

Pfizer monkey virus (MPMV) in several primate colonies known to contain MPMVinfected animals (Fine et al., 19’78b, c; Devare et al., 1978). In particular, rhesus monkeys housed at Yerkes Primate Center were derived from the same colony (U. S. Air Force School of Aerospace Medicine, San Antonio, Tex.) as the female rhesus monkey from which MPMV was originally isolated (Mason et al., 1972). Rhesus monkeys housed at the Litton Bionetics’ colony were maintained in the same primate holding area as rhesus monkeys used in MPMV oncogenicity studies (Fine et al., 1975). Consequently, we previously theorized that precipitating antibody to MPMV in sera of monkeys from these two colonies could thus reflect horizontal transmission from animals known to have prior MPMV infections. In studies described here, antibodies to MPMV were found in macaque sera from five additional primate centers, none of which were known to contain MPMV-infected animals. Antibodies to MPMV were detected at generally equivalent frequency from all seven colonies. This observation coupled with detection of MPMV antibodies in several groups of

58

FINE

AND

ARTHUR

TABLE DISTRIBUTION

OF MMC-1

Colony California Center

Primate (Davis, Calif.)

AND MPMV

3

REACTIVE

Species

M. M. M. M.

mulatta

ANTIBODIES

IN CAPTIVE

PRIMATES

No. animals tested

Grouping

MMC-1

MPMV

16 (11) 0 2 (9) 3 (17)

38 (26) 0 9 (43) 0

radiata arctoides

Normal Lymphoma Normal Normal

mulatta

Normal

24

0

2 (8)

mulatta

144 5 21 18

No. animals positive (% ) against

Delta Regional Primate Center (Covington, La.)

M.

Hazelton Colony Va.)Q

Ceropithecus aethiops M. fascicularis

Normal Normal

17 11

6 (35) 0

3 (18) 1 (9)

Rockefeller University (New York, N. Y.)

M. mulatta

Normal

50

6 (12)

3 (6)

University of Cincinnati Children’s Hospital Research Foundation (Cincinnati, Ohio)

M. mulatta

Normal

7

0

1 (14)b

Yerkes Primate Center (Atlanta, Ga.)

M. mulatta M. mulatta M. mulatta

Normal Irradiated Progeny

4 (10)

3 (7) 2 (8) 2 (15)

M. mulatta M. mulatta M. mulatta

Normal/colony Normal/imported Irradiated

Primate (Springfield,

Litton Bionetics, Inc. Primate Colony (Kensington, Md.)

of irradiated born

42 24 13 83 40 8

2 03) 2 (15)

2 (2) 2 (5) 0

13 (16) 6 (15) 1 (13)

a All animals tested were recently imported; sera were collected while animals were in quarantine. * This serum showed reactivity for both MPMV and SMRV in a confirmatory competition radioimmune assay for lz51-MPMV. MMTV, MuLV, SW, and noninfected A204 cell lysates did not compete for antibody binding of ‘%I-MPMV in this assay.

recently imported monkeys suggests that anti-MPMV response may be against proteins coded by the portion of the MPMV genome endogenous to all Old World monkeys. The data in this report suggest that various macaque species differ in their susceptibility to MPMV infection or expression of the portion of the MPMV genome endogenous to Old World monkeys. For example, at the California Primate Center, Macaca radiata had the highest frequency of MPMV antibody (48%), although no antibody was detected in 18 Macaca arctoides tested. The two macaque

species (Macaca mulatta and Macaca radiata) with the highest frequency of MPMV antibody are native to India. In contrast, the two macaque species having the lowest frequencies of MPMV antibody originate from Southeast Asia; Macaca arctoides originate from Burma and the Siam peninsula and Macaca fascicularis originate from Sumatra and Borneo (Sanderson, 1957). Naturally occurring antibodies to MPMV have been shown here to effectively neutralize MPMV infection. We previously demonstrated that the envelope glycoprotein gp’70 recognizes the cell surface re-

RETROVIRUS

ANTIBODIES TABLE

COMPARISON

OF NEUTRALIZING

AND PRECIPITATING AND NONINOCULATED

IN PRIMATES

59

4 ANTIBODIES TO MPMV Macaca mulatta

IN MPMV-INOCULATED

Antibody

Animal

history

Primate

center

dPMV inoculated Colony born Colony born

Litton Litton

Joninoculated Colony born Imported Imported Imported Colony born

Litton Bionetics, Inc. Litton Bionetics, Inc. Litton Bionetics, Inc. Rockefeller University California Primate Center

Bionetics, Bionetics,

Inc. Inc.

l/Precipitating (RIP) titer”

l/Binding UPA) titer*

l/Neutralizing titerC

6750 3200

+3 +2

160 128

100 800 NT NT NT

NTd NTd +1 (100) +2 (100) +3 (50)

64 64 4 8 <5

’ Titer of serum binding 220% of iZI-MPMV. * Binding titer represents dilution giving positive reaction in IPA assay for hrough +3 indicate intensity of positive reactions, +l being lowest intensity. ’ Based on inhibition of MPMV-induced syncytia formation in KC cells. Data :iving 50% inhibition of syncytia formation compared to control. d Not tested.

eptor in the infection process (Fine et al., 980). Therefore, we speculate that the .eutralizing antibodies are directed at the ~70. Additionally, suggesting that gp70 3 the protein involved is the fact that laEV and SMRV can effectively remove eutralizing activity from MPMV-positive era (data not shown). Since MPMV, BaEV, nd SMRV have common cross-reactive ntigens in their envelope gp70 molecule Devare et al., 1978b; Stephenson et al., 976). Consequently, these naturally ocurring antibodies may play a protective ole against infection from viruses having hared envelope antigens. Our demonstration of naturally occuring antibodies to the endogenous maaque virus allowed us to determine that nere is no preferential expression of an.body to MMC-1 virus in any particul.ar macaque species. Although expression of ntibodies to both MPMV and MMC-1 was bserved in several Macaca mulatta, there ‘as a lack of correlation in the frequency F antibody to the two viruses. This conrms that the natural expression and recynition of MPMV and MMC-1 are con*oiled independently, as was expected for

reactivity

respective

virus.

represent

dilution

Values

+l

of serum

horizontally and genetically transmitted viruses. In addition, although expression of antibodies to MPMV appears to have no correlation with either age or sex, expression of antibodies to the MMC-1 virus appears to be related to both age and sex, as females and older animals had the highest frequency of antibodies to MMC1. Antibody expression relative to age (Arthur and Fine, 1979; Nowinski and Kaehler, 1974) and sex (Arthur and Fine, 1979; Fine et al., 1978a) has been demonstrated for other retrovirus systems. The natural antibody response to MMC1 is directed predominantly against the envelope protein gp73. The envelope protein of murine retroviruses is also the major target antigen for natural antibodies in mouse populations (Arthur et al., 1978; Arthur and Fine, 1979; Ihle and Lazar, 1977; Lee and Ihle, 1975). From these serological studies, we can now conclude that expression of endogenous and horizontally transmitted primate retroviruses is distributed throughout captive populations of Old World monkeys (of 507 animals tested, 129 had antibodies to either MPMV or MMC-l), as

60

FINE

AND

has been previously found for expression of murine and feline retroviruses in their respective host species (Nowinski and Kaehler, 19’74; Essex et al., 1975). ACKNOWLEDGMENTS The technical assistance of R. Imming, and M. Hartsock is acknowledged. We thank for assistance in statistical analysis of work was supported by the Virus Cancer Contract NOl-CO-75380, National Cancer Public Health Service.

J. Kmetz, C. Riggs data. This Program, Institute,

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RETROVIRUS

ANTIBODIES

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