Epstein-Barr Virus Transformation of Saimiri sciureus (Squirrel Monkey) B Cells and Generation of a Plasmodium brasilianum-Specific Monoclonal ...
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INFECTION AND IMMUNITY, JUIY 1991, p. 2285-2290 Vol. 59, No. 7
0019-9567/911072285-06$02.00/0
Copyright C 1991, American Society for Microbiology
Epstein-Barr Virus Transformation of Saimiri sciureus (Squirrel
Monkey) B Cells and Generation of a Plasmodium brasilianum-
Specific Monoclonal Antibody in P. brasilianum-Infected Monkeys
CARLO 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
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The new-world monkeys Saimiri sciureus (squirrel monkeys) are currently used as a model to test the efficacy
of 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 six
healthy 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 with
Plasmodium brasilianum. In the same three animals, the frequency of spleen B cells committed to the production
of P. brasilianum-specific antibody ranged from 1 in 2,211 to 1 in 9,099. One B-lymphoblastoid cell line producing
anti-P. brasilianum-specific antibody was cloned twice, and the immunoglobulin G produced was purified. This
monoclonal antibody recognized a parasite component of 197 kDa and was specific for Plasmodium malaiae and
P. brasilianum parasites. These data document that squirrel monkey B cells naturally primed by an infectious
agent can be efficiently used to produce monospecific antibodies against the infectious agent.
Epstein-Barr virus (EBV) is a herpesvirus that infects squirrel monkey B cells. We addressed this issue by quan-
human B cells (reviewed in reference 27). EBV binds spe- titating in limiting-dilution culture assays (17) the B lym-
cifically to the complement receptor type 2 (CD21) for the phocytes clonally committed to the production of immuno-
C3d fragment of the third component of the complement globulin M (IgM), IgG, and P. brasilianum-specific
expressed at the surface of mature B cells (11). Although immunoglobulin. In addition, the ability of EBV to transform
most B cells are infected by EBV, only a fraction of them are squirrel monkey B cells was used to generate a monoclonal
induced to express viral antigens such as EBNA-1 and then B-LCL-producing antibody specific for a P. brasilianum
to proliferate and produce immunoglobulins in a T cell- component of 197 kDa.
independent fashion (1, 32). EBV-infected B cells can thus
give rise to long-term proliferating lymphoblastoid cell lines
(LCLs). Through appropriate selection of antibodies pro- MATERIALS AND METHODS
duced by B-LCLs, human monoclonal antibodies have been
obtained (3, 4). EBV. The B95-8 marmoset lymphoma cell line (provided
Beside human B cells, EBV has been shown to transform by A. A. Ansari, Emory University, Atlanta, Ga.) was
primate B cells. However, inefficient or no EBV transfor- maintained in RPMI 1640 medium supplemented with 10%
mation of B cells from new-world monkeys, in particular fetal calf serum, 2 mM L-glutamine, 100 U of penicillin per
from Saimiri sciureus (squirrel monkeys), has been reported ml, 100 ,ug of streptomycin per ml, 25 mM N-2-hydroxyeth-
(10, 15, 19). Squirrel monkeys are naturally infected by ylpiperazine-N'-2-ethanesulfonic acid, nonessential amino
Plasmodium brasilianum, which is thought to be a Plasmo- acids (1% of a 10Ox stock solution), 1 mM sodium pyruvate,
dium malariae strain that has recently become adapted in and 5 x 10-5 2-mercaptoethanol (all from GIBCO, Grand
new-world monkeys (7). In addition, squirrel monkeys are Island, N.Y.). This medium is hereafter referred to as
permissive hosts for the human malaria species Plasmodium complete medium (CM). The B95-8 culture supernatant
falciparum and P. vivax (7). As such they have been used in containing living virus, obtained as previously described
recent years to evaluate the efficacy of vaccines designed to (14), was stored at -80°C until use.
induce protective immunity against sporozoites and blood B cell preparation. Blood (3 ml) was drawn from the
stages of these two species (9, 21). The possibility of femoral veins of six healthy adult S. sciureus boliviensis
obtaining monoclonal antibodies (MAbs) efficiently from monkeys (three males and three females) by using heparin-
infected squirrel monkeys could allow the identification of ized Vacutainers. Peripheral blood mononuclear cells
conformationally intact antigenic determinants expressed by (PBMC) were purified by using Ficoll Paque (Pharmacia
living parasites. In addition, passive transfer of homologous Fine Chemicals, Uppsala, Sweden) gradient centrifugation.
antibodies specific for defined parasite antigens could prove After plastic adherence, PBMC were reacted with sheep
useful in evaluating their protective effect. erythrocytes (RBC) previously treated with 2-aminoethyl-
In this study we reassessed the capacity of EBV to infect isothiouronium bromide hydrobromide (Sigma Chemical
Co., St. Louis, Mo.). Rosetting cells were depleted by
gradient centrifugation as described previously (6). Nonro-
setting cells (T cell-depleted PBMC) were used as the source
*
Corresponding author. of B cells. Because of the small number of cells available (0.2
22852286 CHIZZOLINI ET AL. INFECT. IMMUN.
x 106 to 1.3 x 106), no attempt was made to quantify the Determination of frequencies of antibody-producing precur-
actual B cells contained in these cell preparations. sors. After incubation with EBV, enriched spleen B cells
Spleens were surgically removed from three other adult S. were diluted and plated at 2,500, 500, 100, 20, and 4 cells per
sciureus boliviensis naturally infected with P. brasilianum. well in round-bottom 96-well plates in the presence of 0.2 x
Spleen tissue was minced with scissors, and a single-cell 106 irradiated (2,500 rads) normal human PBMC per well as
suspension was obtained by using a homogenizer. Spleen feeder cells. Forty-eight replicates for each cell dose, includ-
cells were incubated in petri dishes (Falcon; Becton Dickin- ing 48 negative controls (feeder cells alone), were seeded.
son, Oxnard, Calif.) for 1 h at 37°C in CM to allow the cells Twenty-one days later, 150 [lI of each culture supernatant
to shed cytophilic immunoglobulin and allow monocytes and was harvested, diluted 1:4 in CM, and tested for the pres-
macrophages to adhere to plastic. Nonadherent cells were ence of IgM, IgG, or P. brasilianum-specific antibodies.
then submitted to two cycles of rosetting with sheep RBC. Positivity for IgM or IgG presence in supernatants was
The nonrosetting cells contained less than 8% sheep RBC defined as an optical density equal to or greater than the
rosette-forming cells and were 24.1, 34.6, and 41.5% positive optical density plus 3 standard deviations of the 48 replicates
for membrane immunoglobulin in three monkeys as deter- of feeder cells (0.05 to 0.1 optical density unit in different
mined by cytofluorometric analysis (FACScan cytofluorom- experiments) as determined in the Falcon assay screening
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eter; Becton Dickinson) with fluorescein isothiocyanate test-ELISA. The fraction of negative wells was then re-
(FITC)-labeled goat anti-S. sciureus IgG (provided by V. corded and utilized to compute the frequency of precursors
Tsang, Centers for Disease Control, Atlanta, Ga.) and FITC- of immunoglobulin-producing cells as described previously
labeled goat anti-mouse immunoglobulin as negative con- (22, 26).
trols. Monoclonal P. brasilianum-specific IgG-producing S. sci-
ELISA titration of IgM and IgG. The Falcon assay screen- ureus cell line. Cells from a culture producing P. brasilianum-
ing test-enzyme-linked immunosorbent assay (ELISA) sys- specific antibody were subcloned twice by limiting dilution.
tem was adopted to determine immunoglobulin titers in The cloned line H12 was then expanded, and the antibody in
culture supernatants by a modification of the procedure of the culture supernatant was purified by precipitation in 50%
Hancock and Tsang (13). In brief, knobs were coated for 150 saturated ammonium sulfate. The antibody contained in
min with 5 ,ug of affinity-purified goat anti-human IgG culture supernatants from the parental cell line or other mass
(gamma chain specific) or anti-human IgM (mu chain spe- cultures was also purified.
cific) (Cappel Laboratories, Organon Teknika Co., West P. brasilianum antigen preparation. Heparinized blood
Chester, Pa.) per ml of phosphate-buffered saline (PBS) (pH from two S. sciureus monkeys infected with P. brasilianum
7.2). Knobs were incubated with appropriate dilutions of (3% parasitemia) was centrifuged. The pellet was suspended
culture supernatants or reference standards for 60 min and in 2 ml of PBS and applied to a column of CF 11 cellulose
then with anti-S. sciureus IgG (H and L chain specific) (Whatman Biosystem Ltd., Maidstone, United Kingdom)
conjugated with horseradish peroxidase for 30 min. The and glass beads (Sigma) previously equilibrated with PBS to
knob-bound enzyme was revealed with the substrate 2,2'- remove the leukocytes and platelets (12, 24). The recovered
azino-di-(3-ethyl-benzthyazoline sulfonate) (Kirkegaard and RBC were fractionated by centrifugation (800 x g for 15 min)
Perry Laboratories, Gaithersburg, Md.), and the A414 was over a discontinuous multistep isotonic Percoll gradient (2).
measured with an automated ELISA reader (Titertek Multi- More than 50% of the RBC in the layer recovered at the
scan; Flow Laboratories, McLean, Va.). Reference binding interface between 40% and 60% Percoll were infected. The
curves were obtained by using purified human IgM or IgG parasites, from young trophozoites to mature schizonts,
(Cappel). The assays for IgM and IgG had a sensitivity of 20 were used as antigen for Western immunoblotting.
ng/ml without evident cross-reactivity up to 20 ,ug/ml. The Western blotting. After two washes in PBS, enriched P.
specificity of this assay for S. sciureus immunoglobulin was brasilianum-infected RBC were extracted in 10 volumes of
verified and confirmed a posteriori when supernatants from lysis buffer (25 mM Tris-HCl [pH 7.6], 50 mM NaCl, 5 mM
B-LCLs producing IgM or IgG were available. EDTA, 1% Triton X-100, 1.5 mM phenylmethylsulfonyl
Immunofluorescence detection of P. brasilianum-specific fluoride, leupeptin [1 ,ug/ml], pepstatin A [1 ,ug/ml]; all from
antibodies. Antigens for the immunofluorescence assay Sigma). Noninfected RBC used as a control were submitted
(IFA) were smears of parasite-infected RBC prepared as to the same procedure. Lysed samples were fractionated
described previously (25). P. brasilianum, P. vivax, and under reducing conditions by sodium dodecyl sulfate-poly-
Plasmodium inui were from infected S. sciureus. Plasmo- acrylamide gel electrophoresis (SDS-PAGE) and transferred
dium cynomolgi was from infected Macaca mulatta. P. to nitrocellulose (0.45-,um pore size; Schleicher and Schuell
malariae was from infected Pan troglodytes. P. falciparum Inc., Keene, N.H.) by using standard procedures (28). After
was from in vitro culture (29). Culture supernatants were transfer, nitrocellulose strips were incubated with purified
incubated on slides for 30 min and then treated sequentially B-LCL antibody (50 jig/ml) overnight at 4°C. After the strips
with FITC-conjugated goat anti-S. sciureus immunoglobulin were washed, they were incubated with horseradish perox-
and with FITC-conjugated swine anti-goat IgG. Slides were idase-conjugated anti-S. sciureus immunoglobulin for 1 h. To
examined on a fluorescence microscope (Olympus; Southern visualize the recognized bands, enzyme activity was re-
Micro Instruments, Inc., Atlanta, Ga.). vealed by using the substrate 3,3-diamino benzidine. Pre-
EBV infection of S. sciureus B cells. T cell-depleted PBMC stained molecular markers (Bethesda Research Laborato-
or enriched spleen B cells (0.2 x 106 to 3 x 106) were ries, Gaithersburg, Md.) were used to determine the relative
incubated overnight at 37°C in 1 ml of freshly thawed EBV. molecular masses.
T cell-depleted PBMC were then cultured in 24-well plates
(Costar, Cambridge, Mass.). Spent medium was replaced RESULTS
every 6 days until vigorous proliferation was evident. Mass
cultures were then maintained in flasks by weekly dilution. Squirrel monkey B cell susceptibility to EBV infection. T
Control cultures not incubated with EBV were similarly cell-depleted PBMC from six healthy squirrel monkeys were
maintained. infected with EBV. From 10 to 15 days later, vigorous cellVOL. 59, 1991 EBV-INDUCED IMMUNOGLOBULIN PRODUCTION IN MONKEYS 2287
TABLE 1. Transformation of squirrel monkey T cell-depleted TABLE 2. Determination of the frequency of squirrel monkey
PBMC by EBV spleen B cell precursors induced by EBV to IgM
and IgG production
No. of Time to
Exposure
Expouratto
Animal
Ammalcells (106) No.l of0' EBVa transformation
(days)b Animala Classb Precursor frequencyc nd Chi square Pe
S1930 IgM 1/1,085 (1/822 to 1/1,432) 4 6.05 60
S120 0.3 + 15 S1939 IgM 1/179 (1/136 to 1/236) 3 0.89 60 IgG 1/51 (1/39 to 1/68) 3 4.11 60 b The supernatant from each microculture was tested for the presence of
IgM and IgG by ELISA.
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a The source of EBV was 1 ml of B95-8 supernatant added to the cell pellet. c Precursor frequencies for IgM- and IgG-secreting B cells were determined
b Day when cell clusters surrounded by lymphoblast were first visible with by the maximum likelihood method. The 95% confidence intervals are given
a phase-contrast inverted microscope. within parentheses.
I
No growth was observed during the 60 days of culture. d Number of cell doses used to compute the precursor frequency. For each
cell dose, 48 replicates were tested; when all 48 replicates were positive or
negative for a given dose, that dose was excluded from the computation.
e Probability of the goodness-of-fit test.
proliferation was observed. By day 18, the growing cells
required subculturing, and continuously growing B-LCLs
were successfully established in all cases. Both IgG and IgM monkeys naturally infected with P. brasilianum were used in
were detected in the supernatants of B-LCLs. No growth a limiting dilution analysis. A representative experiment is
was recorded when T cell-depleted PBMC from four of these shown in Fig. 1. In mass cultures of spleen B cells from the
squirrel monkeys were maintained in culture for 8 weeks three infected animals, the time to transformation was in the
without previous infection with EBV (Table 1). same range of that observed with T cell-depleted PBMC
Frequency analysis of Saimiri B cell precursors committed from normal animals. For three monkeys the frequency of B
to the production of IgM and IgG. The results described cells committed to the production of IgG, ranging from 1 in
above demonstrate that EBV is able to transform Saimiri B 45 to 1 in 66, did not overlap and was higher than the
cells. We were then interested in measuring the frequency of frequency of B cells committed to the production of IgM
B cell precursors activated by EBV to produce IgM or IgG. (ranging from 1 in 179 to 1 in 1,085) (Table 2). Because these
To this aim, enriched spleen B cells from three squirrel monkeys were infected with P. brasilianum, we were also
1 .
co
n 0.5
o 0.3-
F
L\0.3
0
z
0
0
EL 0.1
0
0.
0.05
0 100
200 300 400 500 600
CELLS PLATED PER CULTURE
FIG. 1. Limiting dilution analysis to determine the precursor frequencies of IgM- and IgG-secreting cells from squirrel monkeys in
EBV-stimulated cultures. Forty-eight replicate cultures were started for each cell dose. IgM and IgG concentrations in 21-day-old
supernatants were determined by using an ELISA. The dashed line at 0.37 corresponds to the zero term of the equation according to the
Poisson 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 179
produced IgM. The dotted lines represent the 95% confidence intervals.2288 CHIZZOLINI ET AL. INFECT. IMMUN.
TABLE 3. Determination of the frequency of P. brasilianum-
infected squirrel monkey spleen B cell precursors induced by I-
i r
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 0.14VOL. 59, 1991 EBV-INDUCED IMMUNOGLOBULIN PRODUCTION IN MONKEYS 2289
Moreover, in humans, B cell precursors for IgM production 2. Barnwell, J. W., R. J. Howard, and L. H. Miller. 1982. Altered
have been reported to have higher frequencies than have IgG expression of Plasmodium knowlesi variant antigen on the
B cell precursors. This possibly reflects the higher propor- erythrocyte membrane in splenectomized rhesus monkeys. J.
tion of B cells bearing mu immunoglobulin heavy chains in Immunol. 128:224-226.
PBMC. In fact, EBV has been shown to activate and 3. Casali, P., G. Inghirami, M. Nakamura, T. F. Davies, and A. L.
Notkins. 1986. Human monoclonals from antigen-specific selec-
transform human B cells bearing mu, gamma, or alpha tion of B lymphocytes and transformation by EBV. Science
immunoglobulin heavy chains to produce IgM, IgG, or IgA, 234:476-479.
respectively, with equal efficacy (20). However, a direct 4. Casali, P., M. Nakamura, F. Ginsberg-Fellner, and A. L. Not-
comparison with results obtained with human PBMC may be kins. 1990. Frequency of B cells committed to the production of
inappropriate. In this study, spleen cells and not PBMC were antibodies to insulin in newly diagnosed patients with insulin-
used to perform the limiting dilution analysis. Thus, our dependent diabetes mellitus and generation of high affinity
results might reflect a preferential homing in the spleen of B human monoclonal IgG to insulin. J. Immunol. 144:3741-3747.
cells bearing gamma immunoglobulin heavy chains. In addi- 5. Chizzolini, C., A. Geinoz, M.-H. Kaufmann, and D. SchriJvers.
tion, the spleens were obtained from P. brasilianum-infected 1990. Defective in vitro production of anti-Plasmodium falci-
parum antibodies in some malaria-immune subjects. Cell. Im-
animals. Malaria parasites are potent T cell-dependent B cell munol. 129:151-160.
Downloaded from http://iai.asm.org/ on February 6, 2021 by guest
mitogens (5). Thus, it is possible that B cells with various 6. Chizzolini, C., and L. Perrin. 1986. Antigen-specific and MHC-
degrees of activation were populating the spleen and were restricted Plasmodium falciparum-induced human T lympho-
more susceptible to EBV transformation. Indeed, P. falci- cyte clones. J. Immunol. 137:1022-1028.
parum products have been shown to enhance human lym- 7. Coatney, G. R., W. E. Collins, M. Warren, and P. G. Contacos.
phocyte transformation by EBV (16). 1971. The primate malarias, p. 231-244. U.S. Department of
The quantification of B cells committed to the production Health, Education and Welfare, Bethesda, Md.
of malaria-specific immunoglobulin has been attempted with 8. Cochrane, A. H., Y. Matsumoto, K. K. Kamboj, M. Maracic,
a different methodology (31). In that work, human B cells R. S. Nussenzweig, and M. Aikawa. 1988. Membrane-associated
antigens of blood stages of Plasmodium brasilianum, a quartan
were activated by mutant EL4 thymoma, leading to activa- malaria parasite. Infect. Immun. 56:2080-2088.
tion and differentiation of about 90% of the B cells. The 9. Collins, W. E., R. S. Nussenzweig, W. R. Ballou, T. K. Ruebush
frequencies of P. falciparum-specific B cells in infected II, E. H. Nardin, J. D. Chulay, W. R. Majarian, J. F. Young,
humans reported by these authors (from 0.1 to 1%) are G. F. Wasserman, I. Bathurst, H. L. Gibson, P. J. Barr, S. L.
higher than those against P. brasilianum found in infected Hoffman, S. S. Wasserman, J. R. Broderson, J. C. Skinner,
squirrel monkeys. Besides differences in the experimental P. M. Procell, V. K. Filipski, and C. Wilson. 1989. Immunization
system utilized, it is also possible that the two different of Saimiri sciureus boliviensis with recombinant vaccines based
malaria species had different interactions with the immune on the circumsporozoite protein of Plasmodium vivax. Am. J.
systems of the reciprocal vertebrate hosts, thus explaining Trop. Med. Hyg. 40:455-464.
10. Falk, L., L. Wolfe, F. Deinhardt, J. Paciga, L. Dombos, G.
discrepancies between the results. Klein, W. Henle, and G. Henle. 1974. Epstein-Barr virus:
Finally, we describe here the P. brasilianum antigenic transformation of non-human primate lymphocytes in vitro. Int.
component recognized by a cloned squirrel monkey B-LCL, J. Cancer 13:363-376.
H12. Mouse MAbs have been produced in the past to 11. Fingeroth, J. D., J. J. Weis, T. F. Tedder, J. L. Strominger,
characterize P. brasilianum components (8), but none of P. A. Biro, and D. T. Fearon. 1984. Epstein-Barr virus receptor
these recognized a 197-kDa antigen. Because of its molecu- of human B lymphocytes is the C3d receptor CR2. Proc. Natl.
lar mass, including the presence of minor bands of lower Acad. Sci. USA 81:4510-4514.
molecular mass in the Western blot, and its IFA pattern, the 12. Grant, P. T., and J. C. Fulton. 1957. The catabolism of glucose
antigen described here is highly reminiscent of the P. falci- by strains of Trypanosoma rhodesiense. Biochem. J. 66:242-
parum gp 195, the precursor of the major merozoite surface 250.
13. Hancock, K., and V. W. Tsang. 1986. Development and optimi-
antigen (18). The epitope recognized by H12 MAb appears to zation of the FAST-ELISA for detecting antibodies to Schisto-
be species specific. This MAb did not cross-react with any soma mansoni. J. Immunol. Methods 92:167-176.
other malaria species, with the exception of the closely 14. Inghirami, G., M. Nakamura, J. E. Balow, A. L. Notkins, and P.
related species P. malariae (7). Casali. 1988. Model for studying virus attachment: identification
MAbs produced in mice by the hybridoma technology and quantification of Epstein-Barr virus-binding cells by using
have been extensively used to characterize several cellular biotinylated virus in flow cytometry. J. Virol. 62:2453-2463.
antigens. They have the greatest chance of success, because 15. Ishida, T., and K. Yamamoto. 1987. Survey of nonhuman
repeated immunizations are possible and spleen cells are primates for antibodies reactive with Epstein-Barr virus (EBV)
easily obtained. However, when the immunogen used is antigens and susceptibility of their lymphocytes for immortal-
denatured, as it is with parasites that do not grow in rodents, ization with EBV. J. Med. Primatol. 16:359-371.
16. Kataaha, P. K., C. A. Facer, and E. J. Holborow. 1984.
mouse MAb could fail to detect conformational epitopes that Plasmodium falciparum products enhance human lymphocyte
might have important biological significance. In this in- transformation by Epstein-Barr virus. Clin. Exp. Immunol.
stance, transformation of B lymphocytes from the parasite- 56:371-376.
susceptible host might be useful in generating MAbs from 17. Lefkovits, I., and H. Waldmann. 1979. Limiting dilution analysis
cells involved in the ongoing in vivo immune process. of cells in the immune system, vol. 1, p. 38-140. Cambridge
University Press, Cambridge.
ACKNOWLEDGMENTS 18. Lyon, J. A., R. H. Geller, J. D. Haines, J. D. Chulay, and J. L.
We thank J. M. Roberts for her help with the computer program Weber. 1986. Epitope map and processing scheme for the
for limiting dilution analysis. We also thank P. Wilkins and P. Millet 195,000-dalton surface glycoprotein of Plasmodium falciparum
for their assistance with the Western blot procedure. merozoites deduced from cloned overlapping segments of the
gene. Proc. Natl. Acad. Sci. USA 83:2989-2993.
REFERENCES 19. Miller, G., T. Shope, H. Lisco, D. Stitt, and M. Lipman. 1972.
1. Aman, P., B. Ehlin-Henriksson, and G. Klein. 1984. Epstein- Epstein-Barr virus: transformation, cytopathic changes, and
Barr virus susceptibility of normal human B lymphocyte popu- viral antigens in squirrel monkey and marmoset leukocytes.
lations. J. Exp. Med. 159:208-220. Proc. Natl. Acad. Sci. USA 69:383-387.2290 CHIZZOLINI ET AL. INFECT. IMMUN.
20. Nakamura, M., S. E. Burastero, Y. Ueki, J. W. Larrick, A. L. of immunocompetent cell frequencies. I. Data analysis. J.
Notkins, and P. Casali. 1988. Probing the normal and autoim- Immunol. 126:1614-1619.
mune B cell repertoire with Epstein-Barr virus. Frequency of B 27. Tosato, G. 1987. The Epstein-Barr virus and the immune sys-
cells producing monoreactive high affinity autoantibodies in tem. Adv. Cancer Res. 49:75-126.
patients with Hashimoto's disease and systemic lupus erythem- 28. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic
atosus. J. Immunol. 141:4165-4172. transfer of proteins from polyacrylamide gels to nitrocellulose
21. Perrin, L. H., B. Merkli, M. Loche, C. Chizzolini, J. Smart, and sheets: procedure and some applications. Proc. Natl. Acad. Sci.
R. Richle. 1984. Antimalarial immunity in Saimiri monkeys. USA 76:4350-4354.
Immunization with surface components of asexual blood stages. 29. Trager, W., and J. B. Jensen. 1976. Human malaria parasite in
J. Exp. Med. 160:441-451. continuous culture. Science 193:673-675.
22. Porter, E. H., and R. J. Berry. 1963. The efficient design of 30. Yarchoan, R., G. Tosato, R. M. Blaese, R. M. Simon, and D. L.
transplantable tumor assays. Br. J. Cancer 17:583-595. Nelson. 1983. Limiting dilution analysis of Epstein-Barr virus-
23. Rangan, S. R. S., L. N. Martin, B. E. Bozelka, N. Wang, and induced immunoglobulin production by human B cells. J. Exp.
B. J. Gormus. 1986. Epstein-Barr virus-related herpesvirus Med. 157:1-14.
from a rhesus monkey (Macaca mulatta) with malignant lym- 31. Wen, L., M. Hanvanich, C. Werner-Favre, N. Brouwers, L. H.
phoma. Int. J. Cancer 38:425-432. Perrin, and R. H. Zubler. 1987. Limiting dilution assay for
24. Scheibel, L. W., and J. Miller. 1969. Cytochrome oxidase human B cells based on their activation by mutant EL4 thy-
Downloaded from http://iai.asm.org/ on February 6, 2021 by guest
activity in platelet-free preparation of Plasmodium knowlesi. J. moma cells: total and anti-malaria responder B cell frequencies.
Parasitol. 55:825-829. Eur. J. Immunol. 17:887-892.
25. Sulzer, A. J., and M. Wilson. 1971. The fluorescent antibody test 32. Zerbini, M., and I. Ernberg. 1983. Can Epstein-Barr virus infect
for malaria. Crit. Rev. Clin. Lab. Sci. 2:601-617. and transform all the B-lymphocytes of human cord blood? J.
26. Taswell, C. 1981. Limiting dilution assays for the determination Gen. Virol. 64:539-547.You can also read
