Int. J. Cancer: 7, 375-379 (1971)

ANTIBODY TO HUMAN CELL LINES WITH AND WITHOUT

(EBV) INFECTION IN SERA FROM PATIENTS WITH DIVERSE VIRAL ILLNESSES

ULTRASTRUCTURAL EVIDENCE FOR EPSTEIN-BARR VIRUS

German BELTRAN, James W. NORTHINGTON, Eduardo LEIDERMAN William J. MOGABGAB, and Walter J. STUCKEY

Department of Medicine, Tulane University, School of Medicine and Charity Hospital of Louisiana, New Orleans, La., USA

One hundred and sixteen pairs of acute and convalescent sera from patients with diverse viral illnesses were studied for Epstein-Rarr virus (ERV) antibody by an indirect immunofluorescent technique using as antigen cells of the EB-3 cell line known to harbor EBV particles. Sera of 23 patients with atypical pneumonia associated with Myco plasma pneumoniae were investigated similarly. We found low titers of antibody in 90% of the cases but failed to detect any significant increase of antibody in the con- valescent sera. These results do not support the suggestion that the development of EBV antibody observed in infectious mononucleosis is due to non-spec%c effects such as proliferation of lymphoid cells.

Subsequently, we reacted 31 sera with cells of four continuous cultures established from the buffy coat of patients with various types of leukemia (TU-I through TU-4). After repeated electron microscope examination, cells of these lines are known to be free of EB Vparticles, but harbor a tubular ultrastructure previously described by others in a variety of cell cultures, Although all 31 sera had reactedpositively with ER-3 cells, they failed to react with the TU cells. We interpreted this observation as indicating a lack of antigenic relationship between the tubular structures and EBV.

The EBV was originally observed in con- tinuous cell cultures obtained from patients with Burkitt’s lymphoma (Epstein et al., 1965). Subsequently, several authors noted the develop- ment of antibodies against EBV in infectious mononucleosis (Henle et al., 1968; Neiderman et al., 1968; Evans et al., 1968; Gerber et al., 1968), as well as the presence of high titers of antibody in patients with Burkitt’s lymphoma (Henle and Henle, 3 966) and nasopharyngeal carcinoma (Old et al., 1966). Thus, a possible causal rela- tionship of EBV to these diseases was inferred although a more cautious interpretation was suggested by Glade et al. (1969), who postulated

that multiplication of latent EBV occurred in lymphoproliferative states and caused a non- specific antibody response. Some support for this viewpoint has been provided by the recent demonstration of elevated anti-EBV titers in sera from patients with sarcoidosis (Hirshaut et a[., 1970). Since lymphoid proliferation occurs in rubella, mumps, herpes simplex and other viral infections, we sought for antibodies against EBV in paired acute phase and convalescent sera from patients with various kinds of viral infections. We also included sera from a group of patients with atypical pneumonia associated with Myco- plasma pneumoniae.

Received: December 28, 1970. Present address : Facultad de Medicina, Universidad de Antioquia, Medellin, Colombia, S.A.

375

BELTRAN ET AL.

Subsequently, 31 sera found to contain anti- EBV antibodies were tested with cells from four cell lines established in our laboratory (Table I) and designated TU-1, TU-2, TU-3 and TU-4. These cells harbor submicroscopic tubular structures of undetermined significance (Beltran et al., 1969) (Figure 1). Since some authors described similar elements in cell cultures derived from patients with infectious mononucleosis and a variety of neoplasias and suggested the possi-

TABLE I

TU CELL LINES _ _ _ _ _ ~

Origin Date started Desig- nation

TU-1 Acutemyelomonocyticleukemia October, 1967 TU-2 Acute myeloblastic leukemia March, 1969 TU-3 Acute lymphoblastic leukemia March, 1969 TU-4 Chronic granulocytic leukemia April, 1969

FIGURE 1 Tubular ultrastructures found in association with

the endoplasmic reticulum in cell from TU-1 cell line. Similar ultrastructures were found in the T-2, T-3 and T-4 cell lines. x 69,000.

bility of their being sub-viral structures (Moses, 1969), an antigenic relationship between them and the EBV was sought.

MATERIAL AND METHODS

Paired samples of sera were retrieved from a file collected over a 10-year period from military, industrial and student populations affected by a variety of virus and mycoplasma illnesses. Acute sera had been obtained at the onset of the clinical illness and the convalescent sera 3 to 4 weeks later. A somewhat similar number of samples was selected for each of the organisms represented in the file (Table IT). The diagnosis had been established by serological methods de- scribed in detail elsewhere (Mogabgab, 1968). Briefly, antibodies had been determined in the acute phase and convalescent sera by complement fixation for herpes simplex virus and adenovirus, hemagglutination-inhibition for parainfluenza atrd rubella virus, and neutralization in W138 cell cultures for coxsackie virus and rhinovirus. For Mycoplasma pneumoniae, complement-fixation and/or tetrazolium reduction inhibition neutrali- zation methods had been used. Isolation proce- dures had also been used for coxsackie virus and adenovirus.

Immunofluorescence method

To search for antibodies against EBV the method described by Henle et al. (1966) was followed. As antigen, we utilized EB3 cells from a case of Burkitt’s lymphoma grown as a con- tinuous suspension culture and very kindly provided by Dr. W. Henle, Children’s Hospital of Philadelphia, USA. Herpes-like virus particles were readily detected in these cells. The TU-1

TABLE I1

DISTRIBUTION OF THE PAIRED SERA ACCORDING TO THE ETIOLOGY

Herpes simplex . . . . . . . . . . . 24 Cases Mycoplasma pneumoniae . . . . . . . 23 Cases Adenovirus T4 . . . . . . . . . . . 21 Cases Coxsackie A2 I . . . . . . . . . . . I9 Cases Rhinovirus (Types 1 B, 15,23,29,30,31) . 19 Cases Rubella virus . . . . . . . . . . . . 17 Cases Parainfluenza (Types 1, 2, 3) . . . . . 16 Cases

Total . . . . . . . . . . . . . . . . 139 Cases

376

EBV ANTIBODIES IN PATIENT SERA

through TU-4 cell lines which were free from herpes-like virus particles upon repeated ultra- structural examination were also used. These lines contain ultrastructures regarded by some as possible sub-viral elements (Moses, 1969). Smears from both the EB3 and the TU cells were prepared. The cells were centrifuged and washed twice with phosphate-buffered saline (PBS), smeared on glass slides, and fixed with acetone for 10 min. When not used immediately they were kept at -20" C. Serial dilutions of the test sera were made starting with a 1 :20 dilution. The cells were exposed to the sera in a humid chamber for 30 min at 37" C. After washing with PBS, they were covered with fluorescein-conjugated goat anti-human IgG globulin (Hyland Labora- tories, Inc., Los Angeles, Calif., USA) and incubated for 30 min at 37" C. The cells were then washed with PBS once more, dried in a' mounted with buffered glycerine. The & d G t were made with a Leitz microscope under UV il-

lumination (Osram HBO 200 W lamp). Known positive and negative control sera, kindly pro- vided by Dr. Henle, were included in every batch. The two members of a given pair of sera were examined concomitantly under identical conditions.

RESULTS

The results obtained from testing 139 paired acute phase and convalescent sera using EB3 cells as antigen are presented in Table 111. Antibody was detected in 125 (90%), but in only 16 in- stances was the titer greater than 190. We found only one convalescent serum sample showing a titer two dilutions higher than the acute serum and no instance of a greater increase. On the other hand, the titer in the convalescent sera decreased by two dilutions in five cases. In all others, the antibody remained unchanged or differed only by one dilution from the acute specimen.

TABLE 111

TITRATION OF ACUTE PHASE AND CONVALESCENT SERA IN AN INDIRECT IMMUNOFLUORESCENT TEST WITH EB3 CELLS

H. simplex M . pneunioniae Adenovirus Coxsackie Rhinovirus Rubella

A C A C A C A C A C A C

160 160 320 160 160 160 80 80 80 80 320 160 160 160 160 160 160 160 80 80 80 80 160 320 160 160 160 160 160 160 80 80 80 80 160 160 80 80 80 160 160 160 80 40 80 80 160 160 80 80 80 80 160 80 40 40 80 80 160 160 80 80 80 80 80 160 40 40 80 40 80 160 80 80 80 80 80 80 40 40 40 80 80 40 80 80 80 80 80 40 40 40 40 80 80 40 80 80 80 40 40 40 40 20 40 40 40 80 80 40 40 80 40 40 40 <20 40 40 40 40 80 40 40 80 40 40 20 40 40 40 40 40 40 80 40 40 40 40 20 20 40 40 40 <20 40 80 40 40 40 40 20 <20 20 80 20 40 40 40 40 40 40 40 1 2 0 <20 20 40 20 40 40 40 40 40 40 20 <20 <20 20 20 20 20 40 40 40 40 40 20 <20 <20 20 20 20 20 40 40 40 40 20 40 <20 <20 20 <20 20 20 40 <20 40 20 20 20 <20 t 2 0 <20 t 2 0 20 40 20 20 20 <20 <20 <20 <20 20 20 20 20 20 20 <20 20 <20 <20 20 <20 20

<20 <20 <20 <20 <20 <2O <20 <20 <20 <20

Parainfl.

A C

80 160 80 80 40 80 40 80 40 80 40 40 40 40 40 40 40 20 40 20 40 20 40 <20 20 20 20 <20

<20 <20 <20 <20

Titer expressed as the reciprocal of the highest dilution. Sera with acute phase (A) and convalescent (C) titers of less than 20 (< 20) are considered negative.

377

BELTRAN ET AL.

Thirty-one sera containing antibodies against the EB-3 cells were tested for antibodies against TU cells. These included two control sera of high titer. None of the 31 demonstrated detectable fluorescence.

DISCUSSION

The observed incidence of EBV antibodies agrees well with the data of Henle and Henle (1966) and Gerber and Rosenblum (1968), who found these antibodies in 8590% of adult popu- lations. The lack of antibody increase in the convalescent sera agrees with the experience of previous authors. For instance, Neiderman et al. (1968) found antibodies against EBV in eight acute sera from patients with coxsackie, adeno- virus and myxovirus infection, but did not detect significant titer changes in the convalescent sera. Evans et al. (1968) reported similar results after studying paired samples of sera in 25 cases of varicella, 25 cases of “ tonsillitis ” and in 50 other non-specific acute infectious diseases. In 14 child- ren with EBV antibodies Henle and Henle (1970) did not find significant changes in titers following episodes of “ non-bacterial tonsillitis or pharyn- gitis ”. Furthermore, while three previously nega- tive children developed EBV antibodies, no sero- conversions were seen in 73 others. Presumably some of the illnesses occurring in these groups were of viral etiology, but the authors did not provide etiological information except for the “ non-bacterial ” nature of the episodes. It was speculated that EBV caused the illnesses of the three children that converted from negative to positive. In serial sera collected from a group of children with both “ short and long incubation

period” hepatitis, McCollum et al. (1969) did not detect any developing antibody. None Of 10 children affected in an outbreak of infectious lymphocytosis studied by Blacklow and Kapikian (1970) developed a four-fold or greater antibody rise to EBV. These reports do not support the contention that the appearance of EBV antibody observed in infectious mononucleosis lacks specificity and is merely associated with a lym- pho-proliferative state. The elevated titers found in Burkitt’s lymphoma and nasopharyngeal car- cinoma are more difficult to interpret since no data are available concerning the serologic status of these patients prior to the development of their diseases. The present study includes instances of viral illnesses not previously studied for EBV antibodies such as those associated with rhino- virus and parainfluenza, as well as cases of myco- plasma-associated pneumonia, and supplements previous observations. It also helps to exclude other possible mechanisms of antibody produc- tion in infectious mononucleosis such as the release of a “ suppressed ” EBV in the presence of a new viral infection. The absence of detectable EBV antibodies using the TU cells suggests that there is no relationship between the EBV and the sub-microscopic tubular structures observed in the four TU cell lines as well as in other con- tinuous cell cultures derived from patients with hematological neoplasias and infectious mono- nucleosis.

ACKNOWLEDGEMENTS

This work was supported by the Greater New Orleans Cancer Society, The Perkins Leukemia Fund and USPHS grant 5 T 12 CA 8087.

ANTICORPS DES LIGNEES CELLULAIRES HUMAINES AVEC ET SANS PREUVE ULTRASTRUCTURALE D’INFECTION PAR LE VIRUS

D’EPSTEIN-BARR (VEB) DANS LE SERUM DE SUJETS ATTEINTS DE DIVERSES AFFECTIONS VIRALES

Cent seize paires de strums ‘‘ aigus ” et “ convalescents ” provenant de malades atteints de diverses affections virales ont t t t ttudites pour la recherche de I’anticorps du virus d’Epstein-Barr ( VEB) par une technique d’immunojluorescence indirecte utilisant comme antighe deJ cellules de la lignte EB-3 connue pour contenir des parti- cules de VEB. Les s6ruUms de 23 malades atteints de pneumonie atypique associte ci Mycoplasma pneumoniae ont ttt ttudits de la m2me facon. Nous avons trouvt de faibles titres d’anticorps dans 90 % des cas mais n’avons pu dtceler aucun accroissement important des anticorps dans les skrums de convalescents. Ces rtsultats ne corroborent

378

EBV ANTIBODIES IN PAT ENT SERA

pas I’hypothkse selon laquelle le dtveloppement de I’anticorps anti- VEB observt dans la mononucltose infectieuse serait dii 2 des efets non spe‘cifques tels que laprol$tration de cellules lymphoiiies.

Par la suite, nous avons fait re‘agir 31 strums sur deb cellules de quatre cultures continues que nous avons conJtitutes u partir de la couenne inflammatoire de malades atteints de divers types de leuce‘mie (TU-I h TU-4). Aprks des examens rtpe‘te‘s au microscope tlectronique, nous avons constatt que les cellules de ces ligntes ne contiennent pas de particufes de VEB, mais abritent une ultrastructure tubulaire dtja observe‘e par d’autres spe‘cialistes dans diverses cultures cellulaires. Bien que les 3I se‘rums aient rtagi positivement vis-a-vis des cellules EB-3, ils n’ont pas rPagi uvec les cellules TU. Nous avons interprttt cette observation comme une indication de I’absence de lien antigtnique entre k s structures tirbulaires et le VEB.

REFERENCES

BELTRAN, G., LEIDERMAN, E., STUCKEY, JR., W. J., and MOGABGAB, W. J., Unusual ultrastructural features in a new continuous cell culture from a man with acute leukemia. (Abstract). Clin. Res., 17, 46 (1969).

BLACKLOW, N. R., and KAPIKIAN, A. Z., Serological studies with EB virus in infectious lymphocytosis. Nature (Lond.), 226, 647 (1970).

EPSTEIN, M. A., BARR, Y. M., and ACHONG, B. G., Studies with Burkitt’s lymphoma. Wistar Inst. Sympos. Monogr., 4, 69-82 (1965).

EVANS, A. S., NEIDERMAN, J. C., and MCCOL- LUM, R. W., Sero-epidemiologic studies of infec- tious mononucleosis with E.B. virus. New Engl. J . Med., 279, 1121-1127 (1968).

GERBER, P., HAMRE, D., MOY, R. A., and ROSEN- BLUM, E. N., Infectious mononucleosis: comple- ment-fixing antibodies to herpes-like virus associated with Burkitt’s lymphoma. Science, 161, 173-175 ( I 968).

GERBER, P., and ROSENBLUM, E. N., The incidence of complement-fixing antibodies to herpes-like viruses in man and rhesus monkeys. Proc. SOC. exp. Biol.

GLADE, P. R., HIRSCHHORN, K., and DOUGLAS, S. D., Herpes-like virus. Lancet, 1, 1049-1050 (1969).

HENLE, G., and HENLE, W., Immunofluorescence in cells derived from Burkitt’s lymphoma. J. Buct.,

(N.Y.), 128, 541-546 (1968).

91, 1248-1256 (1966).

HENLE, G., and HENLE, W., Observations on child- hood infections with Epstein-Barr virus. J. infect. Dis., 121, 303-313 (1970).

HENLE, G., HENLE, W., and DIEHL, V., Relation of Burkitt’s tumor-associated herpes-type virus to infectious mononucleosis. Proc. nut. Acad. Sci.

HIRSHAUT, Y., GLADE, P., VIEIRA, L. O., AINBENDER, E., DVOPAK, B., and STILZBACH, L. E., Sarcoidosis. Serological evidence for herpes-like virus infection. New Engl. J. Med., 283, 502-506 (1970).

MCCOLLUM, R. W., NEIDERMAN, J. C., EVANS, A. S., and GILES, J. P., Viruses in viral hepatitis. Lancet,

MOGABGAB, W. J., Acute respiratory illnesses in university (1962-1966), military and industrial (1962-1963) populations. Amer. Rev. resp. Dis.,

MOSES, H. L., The circulating lymphocyte-its role in infectious mononucleosis. (In Clinical Staff Con- ference). Ann. intern. Med., 69, 345-349 (1969).

NEIDERMAN, J. C., MCCOLLUM, R. W., HENLE, G., and HENLE, W., Infectious mononucleosis. Clinical manifestations in relation to E. B. virus antibody. J. Amer. med. Ass., 203, 205-209 (1968).

OLD, L. J., BOYSE, E. A., and OETTGEN, H. F., Precipitating antibody in human serum to an anti- gen present in cultured Burkitt’s lymphoma cells. Pvoc. nut. Acud. Sci. (Wash.), 56, 1699-1704(1966).

(Wash.), 59, 94-98 (1968).

1, 778-779 (1969).

98, 359-379 (1968).

379