Vol. 59, No. 7INFECTION AND IMMUNITY, JUIY 1991, p. 2285-22900019-9567/911072285-06$02.00/0Copyright C 1991, American Society for Microbiology

Epstein-Barr Virus Transformation of Saimiri sciureus (SquirrelMonkey) B Cells and Generation of a Plasmodium brasilianum-

Specific Monoclonal Antibody in P. brasilianum-Infected MonkeysCARLO CHIZZOLINI, ALEXANDER J. SULZER, MELISSA A. OLSEN-RASMUSSEN,

AND WILLIAM E. COLLINS*

Malaria Branch, Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control,Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia 30333

Received 14 January 1991/Accepted 17 April 1991

The new-world monkeys Saimiri sciureus (squirrel monkeys) are currently used as a model to test the efficacyof vaccines against human malaria. To improve our knowledge on this model, we tested the susceptibility of S.sciureus B cells to Epstein-Barr virus (EBV) infection. B-lymphoblastoid cell lines were obtained from six of sixhealthy animals after infection with the B95-8 source of EBV. The frequency distributions of spleen B cells

clonally committed to the production of immunoglobulins M and G, as measured by limiting dilution analysis,were from 1 in 179 to 1 in 1,085 and from 1 in 45 to 1 in 60, respectively, in three monkeys naturally infected withPlasmodium brasilianum. In the same three animals, the frequency of spleen B cells committed to the productionofP. brasilianum-specific antibody ranged from 1 in 2,211 to 1 in 9,099. One B-lymphoblastoid cell line producinganti-P. brasilianum-specific antibody was cloned twice, and the immunoglobulin G produced was purified. Thismonoclonal antibody recognized a parasite component of 197 kDa and was specific for Plasmodium malaiae andP. brasilianum parasites. These data document that squirrel monkey B cells naturally primed by an infectiousagent can be efficiently used to produce monospecific antibodies against the infectious agent.

Epstein-Barr virus (EBV) is a herpesvirus that infectshuman B cells (reviewed in reference 27). EBV binds spe-cifically to the complement receptor type 2 (CD21) for theC3d fragment of the third component of the complementexpressed at the surface of mature B cells (11). Althoughmost B cells are infected by EBV, only a fraction of them areinduced to express viral antigens such as EBNA-1 and thento proliferate and produce immunoglobulins in a T cell-independent fashion (1, 32). EBV-infected B cells can thusgive rise to long-term proliferating lymphoblastoid cell lines(LCLs). Through appropriate selection of antibodies pro-duced by B-LCLs, human monoclonal antibodies have beenobtained (3, 4).

Beside human B cells, EBV has been shown to transformprimate B cells. However, inefficient or no EBV transfor-mation of B cells from new-world monkeys, in particularfrom Saimiri sciureus (squirrel monkeys), has been reported(10, 15, 19). Squirrel monkeys are naturally infected byPlasmodium brasilianum, which is thought to be a Plasmo-dium malariae strain that has recently become adapted innew-world monkeys (7). In addition, squirrel monkeys arepermissive hosts for the human malaria species Plasmodiumfalciparum and P. vivax (7). As such they have been used inrecent years to evaluate the efficacy of vaccines designed toinduce protective immunity against sporozoites and bloodstages of these two species (9, 21). The possibility ofobtaining monoclonal antibodies (MAbs) efficiently frominfected squirrel monkeys could allow the identification ofconformationally intact antigenic determinants expressed byliving parasites. In addition, passive transfer of homologousantibodies specific for defined parasite antigens could proveuseful in evaluating their protective effect.

In this study we reassessed the capacity of EBV to infect

* Corresponding author.

squirrel monkey B cells. We addressed this issue by quan-titating in limiting-dilution culture assays (17) the B lym-phocytes clonally committed to the production of immuno-globulin M (IgM), IgG, and P. brasilianum-specificimmunoglobulin. In addition, the ability of EBV to transformsquirrel monkey B cells was used to generate a monoclonalB-LCL-producing antibody specific for a P. brasilianumcomponent of 197 kDa.

MATERIALS AND METHODS

EBV. The B95-8 marmoset lymphoma cell line (providedby A. A. Ansari, Emory University, Atlanta, Ga.) wasmaintained in RPMI 1640 medium supplemented with 10%fetal calf serum, 2 mM L-glutamine, 100 U of penicillin perml, 100 ,ug of streptomycin per ml, 25 mM N-2-hydroxyeth-ylpiperazine-N'-2-ethanesulfonic acid, nonessential aminoacids (1% of a 10Ox stock solution), 1 mM sodium pyruvate,and 5 x 10-5 2-mercaptoethanol (all from GIBCO, GrandIsland, N.Y.). This medium is hereafter referred to ascomplete medium (CM). The B95-8 culture supernatantcontaining living virus, obtained as previously described(14), was stored at -80°C until use.B cell preparation. Blood (3 ml) was drawn from the

femoral veins of six healthy adult S. sciureus boliviensismonkeys (three males and three females) by using heparin-ized Vacutainers. Peripheral blood mononuclear cells(PBMC) were purified by using Ficoll Paque (PharmaciaFine Chemicals, Uppsala, Sweden) gradient centrifugation.After plastic adherence, PBMC were reacted with sheeperythrocytes (RBC) previously treated with 2-aminoethyl-isothiouronium bromide hydrobromide (Sigma ChemicalCo., St. Louis, Mo.). Rosetting cells were depleted bygradient centrifugation as described previously (6). Nonro-setting cells (T cell-depleted PBMC) were used as the sourceof B cells. Because of the small number of cells available (0.2

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x 106 to 1.3 x 106), no attempt was made to quantify theactual B cells contained in these cell preparations.

Spleens were surgically removed from three other adult S.sciureus boliviensis naturally infected with P. brasilianum.Spleen tissue was minced with scissors, and a single-cellsuspension was obtained by using a homogenizer. Spleencells were incubated in petri dishes (Falcon; Becton Dickin-son, Oxnard, Calif.) for 1 h at 37°C in CM to allow the cellsto shed cytophilic immunoglobulin and allow monocytes andmacrophages to adhere to plastic. Nonadherent cells werethen submitted to two cycles of rosetting with sheep RBC.The nonrosetting cells contained less than 8% sheep RBCrosette-forming cells and were 24.1, 34.6, and 41.5% positivefor membrane immunoglobulin in three monkeys as deter-mined by cytofluorometric analysis (FACScan cytofluorom-eter; Becton Dickinson) with fluorescein isothiocyanate(FITC)-labeled goat anti-S. sciureus IgG (provided by V.Tsang, Centers for Disease Control, Atlanta, Ga.) and FITC-labeled goat anti-mouse immunoglobulin as negative con-trols.ELISA titration of IgM and IgG. The Falcon assay screen-

ing test-enzyme-linked immunosorbent assay (ELISA) sys-tem was adopted to determine immunoglobulin titers inculture supernatants by a modification of the procedure ofHancock and Tsang (13). In brief, knobs were coated for 150min with 5 ,ug of affinity-purified goat anti-human IgG(gamma chain specific) or anti-human IgM (mu chain spe-cific) (Cappel Laboratories, Organon Teknika Co., WestChester, Pa.) per ml of phosphate-buffered saline (PBS) (pH7.2). Knobs were incubated with appropriate dilutions ofculture supernatants or reference standards for 60 min andthen with anti-S. sciureus IgG (H and L chain specific)conjugated with horseradish peroxidase for 30 min. Theknob-bound enzyme was revealed with the substrate 2,2'-azino-di-(3-ethyl-benzthyazoline sulfonate) (Kirkegaard andPerry Laboratories, Gaithersburg, Md.), and the A414 wasmeasured with an automated ELISA reader (Titertek Multi-scan; Flow Laboratories, McLean, Va.). Reference bindingcurves were obtained by using purified human IgM or IgG(Cappel). The assays for IgM and IgG had a sensitivity of 20ng/ml without evident cross-reactivity up to 20 ,ug/ml. Thespecificity of this assay for S. sciureus immunoglobulin wasverified and confirmed a posteriori when supernatants fromB-LCLs producing IgM or IgG were available.Immunofluorescence detection of P. brasilianum-specific

antibodies. Antigens for the immunofluorescence assay(IFA) were smears of parasite-infected RBC prepared asdescribed previously (25). P. brasilianum, P. vivax, andPlasmodium inui were from infected S. sciureus. Plasmo-dium cynomolgi was from infected Macaca mulatta. P.malariae was from infected Pan troglodytes. P. falciparumwas from in vitro culture (29). Culture supernatants wereincubated on slides for 30 min and then treated sequentiallywith FITC-conjugated goat anti-S. sciureus immunoglobulinand with FITC-conjugated swine anti-goat IgG. Slides wereexamined on a fluorescence microscope (Olympus; SouthernMicro Instruments, Inc., Atlanta, Ga.).EBV infection of S. sciureus B cells. T cell-depleted PBMC

or enriched spleen B cells (0.2 x 106 to 3 x 106) wereincubated overnight at 37°C in 1 ml of freshly thawed EBV.T cell-depleted PBMC were then cultured in 24-well plates(Costar, Cambridge, Mass.). Spent medium was replacedevery 6 days until vigorous proliferation was evident. Masscultures were then maintained in flasks by weekly dilution.Control cultures not incubated with EBV were similarlymaintained.

Determination of frequencies of antibody-producing precur-sors. After incubation with EBV, enriched spleen B cellswere diluted and plated at 2,500, 500, 100, 20, and 4 cells perwell in round-bottom 96-well plates in the presence of 0.2 x106 irradiated (2,500 rads) normal human PBMC per well asfeeder cells. Forty-eight replicates for each cell dose, includ-ing 48 negative controls (feeder cells alone), were seeded.Twenty-one days later, 150 [lI of each culture supernatantwas harvested, diluted 1:4 in CM, and tested for the pres-ence of IgM, IgG, or P. brasilianum-specific antibodies.Positivity for IgM or IgG presence in supernatants wasdefined as an optical density equal to or greater than theoptical density plus 3 standard deviations of the 48 replicatesof feeder cells (0.05 to 0.1 optical density unit in differentexperiments) as determined in the Falcon assay screeningtest-ELISA. The fraction of negative wells was then re-corded and utilized to compute the frequency of precursorsof immunoglobulin-producing cells as described previously(22, 26).Monoclonal P. brasilianum-specific IgG-producing S. sci-

ureus cell line. Cells from a culture producing P. brasilianum-specific antibody were subcloned twice by limiting dilution.The cloned line H12 was then expanded, and the antibody inthe culture supernatant was purified by precipitation in 50%saturated ammonium sulfate. The antibody contained inculture supernatants from the parental cell line or other masscultures was also purified.

P. brasilianum antigen preparation. Heparinized bloodfrom two S. sciureus monkeys infected with P. brasilianum(3% parasitemia) was centrifuged. The pellet was suspendedin 2 ml of PBS and applied to a column of CF 11 cellulose(Whatman Biosystem Ltd., Maidstone, United Kingdom)and glass beads (Sigma) previously equilibrated with PBS toremove the leukocytes and platelets (12, 24). The recoveredRBC were fractionated by centrifugation (800 x g for 15 min)over a discontinuous multistep isotonic Percoll gradient (2).More than 50% of the RBC in the layer recovered at theinterface between 40% and 60% Percoll were infected. Theparasites, from young trophozoites to mature schizonts,were used as antigen for Western immunoblotting.

Western blotting. After two washes in PBS, enriched P.brasilianum-infected RBC were extracted in 10 volumes oflysis buffer (25 mM Tris-HCl [pH 7.6], 50 mM NaCl, 5 mMEDTA, 1% Triton X-100, 1.5 mM phenylmethylsulfonylfluoride, leupeptin [1 ,ug/ml], pepstatin A [1 ,ug/ml]; all fromSigma). Noninfected RBC used as a control were submittedto the same procedure. Lysed samples were fractionatedunder reducing conditions by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and transferredto nitrocellulose (0.45-,um pore size; Schleicher and SchuellInc., Keene, N.H.) by using standard procedures (28). Aftertransfer, nitrocellulose strips were incubated with purifiedB-LCL antibody (50 jig/ml) overnight at 4°C. After the stripswere washed, they were incubated with horseradish perox-idase-conjugated anti-S. sciureus immunoglobulin for 1 h. Tovisualize the recognized bands, enzyme activity was re-vealed by using the substrate 3,3-diamino benzidine. Pre-stained molecular markers (Bethesda Research Laborato-ries, Gaithersburg, Md.) were used to determine the relativemolecular masses.

RESULTS

Squirrel monkey B cell susceptibility to EBV infection. Tcell-depleted PBMC from six healthy squirrel monkeys wereinfected with EBV. From 10 to 15 days later, vigorous cell

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TABLE 1. Transformation of squirrel monkey T cell-depletedPBMC by EBV

No. of Exposure to Time toAnimal No.l of0' Expourat transformationAmmalcells (106) EBVa (days)b

SIlo 0.35 + 15SIlo 0.35 - >60cSIll 0.6 + 11S114 0.35 + 11S114 0.35 - >60S120 0.3 + 15S120 0.3 - >60S164 0.2 + 15S165 0.25 + 11S165 0.25 - >60

a The source of EBV was 1 ml of B95-8 supernatant added to the cell pellet.b Day when cell clusters surrounded by lymphoblast were first visible with

a phase-contrast inverted microscope.I No growth was observed during the 60 days of culture.

proliferation was observed. By day 18, the growing cellsrequired subculturing, and continuously growing B-LCLswere successfully established in all cases. Both IgG and IgMwere detected in the supernatants of B-LCLs. No growthwas recorded when T cell-depleted PBMC from four of thesesquirrel monkeys were maintained in culture for 8 weekswithout previous infection with EBV (Table 1).Frequency analysis of Saimiri B cell precursors committed

to the production of IgM and IgG. The results describedabove demonstrate that EBV is able to transform Saimiri Bcells. We were then interested in measuring the frequency ofB cell precursors activated by EBV to produce IgM or IgG.To this aim, enriched spleen B cells from three squirrel

1 .

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TABLE 2. Determination of the frequency of squirrel monkeyspleen B cell precursors induced by EBV to IgM

and IgG production

Animala Classb Precursor frequencyc nd Chi square Pe

S1930 IgM 1/1,085 (1/822 to 1/1,432) 4 6.05 <0.05IgG 1/45 (1/34 to 1/60) 3 3.21 <0.05

S1938 IgM 1/1,005 (1/763 to 1/1,383) 4 7.73 <0.05IgG 1/60 (1/50 to 1/88) 3 6.77 >0.05

S1939 IgM 1/179 (1/136 to 1/236) 3 0.89 <0.05IgG 1/51 (1/39 to 1/68) 3 4.11 <0.05

a The spleen donors were infected with P. brasilianum.b The supernatant from each microculture was tested for the presence of

IgM and IgG by ELISA.c Precursor frequencies for IgM- and IgG-secreting B cells were determined

by the maximum likelihood method. The 95% confidence intervals are givenwithin parentheses.

d Number of cell doses used to compute the precursor frequency. For eachcell dose, 48 replicates were tested; when all 48 replicates were positive ornegative for a given dose, that dose was excluded from the computation.

e Probability of the goodness-of-fit test.

monkeys naturally infected with P. brasilianum were used ina limiting dilution analysis. A representative experiment isshown in Fig. 1. In mass cultures of spleen B cells from thethree infected animals, the time to transformation was in thesame range of that observed with T cell-depleted PBMCfrom normal animals. For three monkeys the frequency of Bcells committed to the production of IgG, ranging from 1 in45 to 1 in 66, did not overlap and was higher than thefrequency of B cells committed to the production of IgM(ranging from 1 in 179 to 1 in 1,085) (Table 2). Because thesemonkeys were infected with P. brasilianum, we were also

0 100 200 300 400 500CELLS PLATED PER CULTURE

600

FIG. 1. Limiting dilution analysis to determine the precursor frequencies of IgM- and IgG-secreting cells from squirrel monkeys inEBV-stimulated cultures. Forty-eight replicate cultures were started for each cell dose. IgM and IgG concentrations in 21-day-oldsupernatants were determined by using an ELISA. The dashed line at 0.37 corresponds to the zero term of the equation according to thePoisson distribution when one B cell precursor is seeded in each well. In this experiment, 1 in 51 added B cells produced IgG and 1 in 179produced IgM. The dotted lines represent the 95% confidence intervals.

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TABLE 3. Determination of the frequency of P. brasilianum-infected squirrel monkey spleen B cell precursors induced by

EBV to P. brasilianum-specific antibody production

Animal Precursor frequencya n Chi square P

SI930 1/9,099 (1/5,501 to 1/15,038) 2 0.14 <0.05S1938 1/2,211 (1/1,642 to 1/2,978) 3 3.59 <0.05SI939 1/4,959 (1/3,336 to 1/7,373) 3 1.71 <0.05a The supematant from each microculture was tested for the presence of

P. brasilianum-specific antibody by the IFA. For further explanation see thefootnotes to Table 2.

interested in evaluating the progenitor frequency of B cellsproducing P. brasilianum-specific antibodies. Parasite-spe-cific antibodies were detected by IFA in cultures from thethree monkeys, with frequencies ranging from 1 in 2,211 to 1in 9,099 (Table 3).

Generation and characterization of a P. brasilianum-specificMAb. Cells from a culture seeded at 2,500 cells and positivefor the presence of IgG and parasite-specific antibodies were

submitted to sequential cloning by limiting dilution first at0.4 cell per well and then at 10 cells per well because lessthan 10% of the wells were growing. The quantitation of IgGin culture fluid of the cloned H12 B-LCL tested at the end of7 days of subculture was consistently above 5 mg/ml in fourdistinct determinations. The H12 IgG recognized P. brasil-ianum and P. malariae asexual blood stages at similarconcentration by the IFA but did not react when testedagainst P. falciparum, P. vivax, P. cynomolgi, and P. inui.The immunofluorescence reaction pattern was diffuse or

reticulated with increasing intensity from late trophozoitesto mature schizonts. In the segmenting cells, the patternappeared to delineate individual merozoites (Fig. 2). ByWestern blot the H12 MAb recognized a major band of 197kDa specifically on parasite-infected RBC but not on nonin-fected RBC. Minor bands were also recognized correspond-ing to 183, 160, 136, 95, 82, and 43 kDa. No bands were

recognized by purified immunoglobulin from an unclonedB-LCL obtained from a healthy animal (Fig. 3).

DISCUSSION

The present study was undertaken to determine whetherEBV is able to infect and transform B cells from squirrelmonkeys, a new-world primate species. The results showthat (i) B cells from healthy or malaria-infected squirrelmonkeys were efficiently transformed by EBV; (ii) thefrequency of spleen B cell precursors transformed by EBVand committed to IgG production was higher than that

FIG. 2. Immunofluorescence staining pattern of MAb H12 withP. brasilianum-infected squirrel monkey RBC.

45 -

9

FIG. 3. Western blot analysis. Percoll-enriched P. brasilianum-infected RBC from squirrel monkeys were lysed, subjected toSDS-PAGE, and transferred to nitrocellulose (lanes A and B).Noninfected RBC were submitted to the same procedure (lane C).Lanes A and C were probed with MAb H12; lane B was probed withpolyclonal antibody from the SIll B-LCL. The numbers to the leftindicate the relative molecular masses in kilodaltons.

committed to IgM production; (iii) through the selection andsubcloning of a B-LCL secreting an antibody of interestingspecificity, relatively large amounts of squirrel monkeymonoclonal IgG were easily obtained; and (iv) partial bio-chemical and structural characterization of a P. brasilianumantigenic component was performed with the H12 squirrelmonkey MAb.

Beside human B cells, EBV and EBV-like viruses havebeen shown to infect and transform B cells from apes andold-world monkeys (10, 15, 19, 23). In one study on 16animals of seven new-world species, including squirrel mon-keys, negative results were reported (15). However, inagreement with Falk et al. (10) and with Miller et al. (19), wefound that squirrel monkey B cells are transformed by theB95-8 source of EBV. That exogenous EBV but not endog-enous virus transformed Saimiri B cells in our assays issupported by the lack of spontaneous growth observed incultures of B cells not incubated with B95-8 supernatants.The 100% rate of successful B cell transformation withsquirrel monkeys in this study is higher than the 20 to 30%success rates previously reported (10, 19). However, tech-nical factors such as EBV preparation and T-cell depletionbefore the EBV infection of B cells may explain this differ-ence.Of interest is the high frequency of EBV-transformed

squirrel monkey B cells. Indeed, the measured frequenciesfor IgG B cell precursors (1 in 45 to 1 in 66) or IgM B cellprecursors (1 in 179 to 1 in 1,085) are possibly underes-timates. In fact, the starting population was composed ofenriched but not pure B cells, and no attempts were made tocorrect the results for the number of actual B cells plated.Although the frequency of EBV-transformed B cells re-

ported in the literature for humans differs according to theparticular protocol followed, the number of B cells commit-ted to IgG production in humans as detected by the meth-odology applied here (30) was from 1 in 145 to 1 in 450, i.e.,2 to 10 times lower than that measured for squirrel monkeys.

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Moreover, in humans, B cell precursors for IgM productionhave been reported to have higher frequencies than have IgGB cell precursors. This possibly reflects the higher propor-tion of B cells bearing mu immunoglobulin heavy chains inPBMC. In fact, EBV has been shown to activate andtransform human B cells bearing mu, gamma, or alphaimmunoglobulin heavy chains to produce IgM, IgG, or IgA,respectively, with equal efficacy (20). However, a directcomparison with results obtained with human PBMC may beinappropriate. In this study, spleen cells and not PBMC wereused to perform the limiting dilution analysis. Thus, ourresults might reflect a preferential homing in the spleen of Bcells bearing gamma immunoglobulin heavy chains. In addi-tion, the spleens were obtained from P. brasilianum-infectedanimals. Malaria parasites are potent T cell-dependent B cellmitogens (5). Thus, it is possible that B cells with variousdegrees of activation were populating the spleen and weremore susceptible to EBV transformation. Indeed, P. falci-parum products have been shown to enhance human lym-phocyte transformation by EBV (16).The quantification of B cells committed to the production

of malaria-specific immunoglobulin has been attempted witha different methodology (31). In that work, human B cellswere activated by mutant EL4 thymoma, leading to activa-tion and differentiation of about 90% of the B cells. Thefrequencies of P. falciparum-specific B cells in infectedhumans reported by these authors (from 0.1 to 1%) arehigher than those against P. brasilianum found in infectedsquirrel monkeys. Besides differences in the experimentalsystem utilized, it is also possible that the two differentmalaria species had different interactions with the immunesystems of the reciprocal vertebrate hosts, thus explainingdiscrepancies between the results.

Finally, we describe here the P. brasilianum antigeniccomponent recognized by a cloned squirrel monkey B-LCL,H12. Mouse MAbs have been produced in the past tocharacterize P. brasilianum components (8), but none ofthese recognized a 197-kDa antigen. Because of its molecu-lar mass, including the presence of minor bands of lowermolecular mass in the Western blot, and its IFA pattern, theantigen described here is highly reminiscent of the P. falci-parum gp 195, the precursor of the major merozoite surfaceantigen (18). The epitope recognized by H12 MAb appears tobe species specific. This MAb did not cross-react with anyother malaria species, with the exception of the closelyrelated species P. malariae (7).MAbs produced in mice by the hybridoma technology

have been extensively used to characterize several cellularantigens. They have the greatest chance of success, becauserepeated immunizations are possible and spleen cells areeasily obtained. However, when the immunogen used isdenatured, as it is with parasites that do not grow in rodents,mouse MAb could fail to detect conformational epitopes thatmight have important biological significance. In this in-stance, transformation of B lymphocytes from the parasite-susceptible host might be useful in generating MAbs fromcells involved in the ongoing in vivo immune process.

ACKNOWLEDGMENTSWe thank J. M. Roberts for her help with the computer program

for limiting dilution analysis. We also thank P. Wilkins and P. Milletfor their assistance with the Western blot procedure.

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