INFECTION AND IMMUNITY, Mar. 1992, p. 1258-1260 0019-9567/92/031258-03$02.00/0

Vol. 60, No. 3

Generation of Human Monoclonal Antibodies That Confer Protection against Pertussis Toxin M.




Laboratorio di Biologia Cellulare, Dipartimento di Genetica, Biologia e Chimica Medical and Dipartmento di Scienze Biomediche e Oncologia Umana, Universit,a di Torino, and Centro di Immunogenetica ed Istocompatibilita, Consiglio Nazionale delle Ricerche,4 10126 Turin, and Sclavo Research Center, 53100 Siena,2 Italy Received 9 October 1991/Accepted 2 January 1992

A panel of human monoclonal antibodies reactive with pertussis toxin has been generated by means of Epstein-Barr virus infection. One of these, the 3F11 monoclonal antibody, showed the ability to neutralize in vitro and in vivo the toxic effects of the toxin. Western blot (immunoblot) analysis located the 3F11 epitope on the S3 subunit.

Pertussis toxin (PT) is reported to include two functionally different moieties. Moiety A includes the enzymatically active subunit Si, while moiety B (subunits S2 to S5) is responsible for binding of PT to the surface of eukaryotic cells and for facilitating the translocation of S1 across the membrane (14). Several murine monoclonal antibodies (MAb) against the different PT subunits have been produced in recent years and used either to define the main structural and functional features of PT (11, 13) or to provide a detailed map of the relevant epitopes involved in toxin neutralization (2, 12). The data gathered in these collaborative efforts have provided the starting point for the development of new and safer vaccines against whooping cough (7, 8). To dissect the serologic response to PT in a human model, we generated a panel of human MAb derived from immunized donors after infection of their circulating B lymphocytes with Epstein-Barr virus (EBV). One of these, the MAb 3F11, featured a strong and reproducible ability to bind the toxin and was associated with a powerful neutralizing activity. The specific epitope of the antibody was located in the S3 subunit of the toxin, a finding consistent with the observations of PT function (6). Heparinized peripheral blood samples were obtained from healthy volunteers injected parenterally with the PT-9K/ 129G nontoxic mutant (8), using a previously reported vaccination schedule (9). Cells were infected with EBV by incubating the nonrosetting peripheral blood mononuclear cells in cell-free supernatants of the B95.8 line (10). Infected cells were then dispersed in 96-well flat-bottom microtiter plates (Nunc, Kamstrup, Denmark; cell range, 10 to 10,000 cells per well) in RPMI 1640 medium supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (HyClone, Logan, Utah). The screening of the specific immunoglobulin (Ig) secretion by EBV lymphoblastoids by an enzyme-linked immunosorbent assay (ELISA) allowed us to identify 25 EBVtransformed cultures secreting detectable amounts of Ig specifically reactive with PT. Quantitative analysis indicated that the average Ig production of the cultures and clones studied was in the range of 0.1 to 10 ,ug/ml. Culture of the cells in 20 U of recombinant interleukin-6 (kindly provided *

by Genetics Institute, Cambridge, Mass.) did not significantly improve the Ig production and secretion of the cell lines. To overcome the low level of cloning efficiency of the lymphoblastoid cells, the cells were cloned three times at 10 to 1,000 cells per well. All of these cell lines were stable with respect to antibody production for up to 18 months. Out of this panel of PT-specific Igs, the human MAb 3F11 featured performance superior to those of the companion cultures and was selected as the prototype human MAb. The Ig secreted by the 3F11 line underwent a two-step purification approach. The culture supernatants (600 ml) were passed on a protein G column (Pharmacia). To separate the bovine IgG contained in the culture medium from the human IgG, the protein G-purified Igs were subjected to hydroxylapatite (Bio-Rad, Richmond, Calif.) chromatography (3). This purification approach made it possible to drastically reduce the contaminating bovine IgG (Fig. 1, lane C). The isotype of the 3F11 antibody was IgGl (K), and the fact that it was monoclonal was confirmed by isoelectric







A 1






FIG. 1. Optical density profile of the material eluted from the hydroxylapatite column. SDS gel electrophoresis patterns of peaks 1 and 2 (left panel) are represented in lanes B and C, respectively. Lane E shows the electrophoretic pattern of the material eluted from the protein G column. Protein G-purified bovine and human Ig (lanes A and D, respectively) were used as controls.

Corresponding author. 1258


VOL. 60, 1992


TABLE 2. Neutralization of PT-induced leukocytosis by human MAb 3F11 treatment

T1.6 8.20




4.65 2.



immunoblot of the human MAb

and of two uncloned EBV lines












control. Numbers

Fetal calf on







focusing (1) (Fig. 2). 3F11




constant of the human MAb

1011 M' (5).

The functional features of the tested in terms of the



MAb 3F11


to neutralize the toxic effects of

PT both in vitro and in vivo. As shown in Table

1, 3F11


the human MAb which in vitro had the

highest neutralizing titer in the PT cytotoxicity test, routinely performed on Chinese hamster ovary (CHO) cells (4). The protective effect was deduced by the ability of the supernatant dilutions of selected cultures to inhibit the clustering of CHO cells (104 cells per well) induced by the addition of PT (80 pg/well). Appropriate dilutions of a standard polyclonal human anti-PT serum were used as a positive control. Indeed, 3 p.g of the human MAb 3F11 per ml


able to neutralize the toxic

effect of PT. The ability toxicity of PT

of the in vivo








by a leukocytosis assay. Female BALB/c mice (5 to 8 weeks old) were injected on day 0 with 0.2 ml of phosphate-buffered saline (PBS) alone or containing 25, 50, or 100 p.g of the human MAb 3F11. Each sample included five mice. On day 1, the mice were injected intraperitoneally with 0.2 ml of PBS containing 0.5 p.g of wild-type PT. On day 5, 5 pl1 of blood was collected from the tails and the total number of leukocytes was counted on a Coulter Counter (Coulter Electronics, Hialeah, Fla.). The was

No. of leukocytes/

Day 0



PBS PBS MAb (100) MAb (50) MAb (25)


7.62 + 0.74 15.87 t 0.58 8.00 t 0.38






t t

0.41 0.56

a Female BALB/c mice were each injected intraperitoneally with 0.2 ml of PBS alone (PBS) or containing human MAb 3F11 (MAb; amount in parentheses is in micrograms) and 0.5 ±Lg of wild-type PT (PT). b Values are leukocyte counts + standard deviations.




3F11 was followed by protection of the mice from the toxic activity of 0.5 ,ug of PT. The protection was dose dependent. Purified B oligomer or Si subunit, immobilized on microtiter plates, was used to study the antigenic specificity of the human MAb 3F11. A specific ELISA indicated that the antibody 3F11 reacted only with the B oligomer. This initial finding has been supported and strengthened in greater detail by running the different subunits on a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis gel (which allows a discrimination of the chains according to the pattern of migration) and further by performing a Western blot (immunoblot) test. As shown in Fig. 3, MAb 3F11 reacted only to subunit S3. This result was also confirmed with recombinant PT subunits expressed in Escherichia coli (8) (data not shown). This work describes the production, characterization, and epitope mapping of a human MAb to PT. We used the technique of EBV transformation of immune B cells, a dependable approach that constantly yields an immortalization of clonal B-cell populations stably secreting IgG reactive with PT. One of these antibodies, the human MAb 3F11, yielded a strong protective effect in vitro and identified a determinant expressed within the S3 subunit of PT. This result suggested that neutralizing activity may be due to the ability of MAb 3F11 to interfere with the binding of PT to the surface of eukaryotic cells and/or with the subsequent access of the Si subunit to its intracellular substrate and the subsequent toxic effect. Antibodies generated through the procedure described above can be used to analyze the complex process of PT



data in Table 2 show that treatment with the human MAb




3F11 Positive control


In vitro neutralization of PT toxic effect



Human MAb Human MAb Human MAb Polyclonal human


40 tg/ml Nonprotective 3 ,ug/ml 1/4,000

Values are MAb concentration or dilution (for polyclonal antibodies) resulting in a total inhibition of the toxic effects induced by 3.2 ng of PT per ml in the CHO cell test (see text). a



S2 -_ S3




FIG. 3. Shown are SDS gel (lane A) of PT stained with Coomassie blue and Western blot (lane B) with MAb 3F11, whose reactivity to the subunit S3 was confirmed.



activity and to understand its pathogenic mechanism in humans. They are ideal tools for defining the minimal antigenic structures that might form a safer synthetic or recombinant vaccine. The results of this study may also find application in overcoming some of the population's reluctance to accept vaccination against whooping cough, fearing adverse reactions that resulted from reagents of the past generation. Furthermore, the neutralizing activity and the high affinity displayed by the human MAb 3F11 appear more than encouraging. The next step will be to direct human MAb technology, mainly in its more recent developments, to the production of reagents to be used in prophylaxis and therapy for patients. We thank L. Nencioni and F. Zappalorto for the protection studies, M. Bugnoli for the Western blotting, and M. Mariani for affinity constant determination. The work was supported by the special project Biotecnologie e Biosensori (Consiglio Nazionale delle Ricerche, Rome, Italy), by the AIDS Project (Istituto Superiore di Sanita, Rome, Italy), and by the Italian Association for Cancer Research (AIRC, Milan, Italy).

REFERENCES 1. Alessio, M., S. Roggero, A. Funaro, L. De Monte, L. Peruzzi, M. Geuna, and F. Malavasi. 1990. CD38 molecule: structural and biochemical analysis on human T lymphocytes, thymocytes and plasma cells. J. Immunol. 145:878-884. 2. Bigio, M., R. Rossi, D. Nucci, M. G. Borri, G. Antoni, A. Bartoloni, and R. Rappuoli. 1988. Monoclonal antibodies against pertussis toxin subunits. FEMS Microbiol. Lett. 51:7-11. 3. De Monte, L., P. Nistico, R. Tecce, P. Dellabona, M. Momo, A. Anichini, M. Mariani, P. G. Natali, and F. Malavasi. 1990. Gene transfer by retrovirus-derived shuttle vectors in the generation of murine bispecific monoclonal antibodies. Proc. Natl. Acad. Sci. USA 87:2941-2945. 4. Hewlett, E. L., K. T. Sauer, G. A. Myers, J. L. Cowell, and R. L. Guerrant. 1983. Induction of a novel morphological response in Chinese hamster ovary cells by pertussis toxin. Infect. Immun. 40:1198-1203.

INFECT. IMMUN. 5. Mariani, M., L. Bracci, R. Presentini, D. Nucci, P. Neri, and G. Antoni. 1987. Immunogenicity of a free synthetic peptide: carrier conjugation enhances antibody affinity for the native protein. Mol. Immunol. 24:297-303. 6. Montecucco, C., M. Tomasi, G. Schiavo, and R. Rappuoli. 1986. Hydrophobic photolabelling of pertussis toxin subunits interacting with lipids. FEBS Lett. 194:301-304. 7. Nicosia, A., M. Perugini, C. Franzini, M. C. Casagli, M. G. Borri, M. Antoni, P. Neri, G. Ratti, and R. Rappuoli. 1986. Cloning and sequencing of pertussis toxin genes: operon structure and gene duplication. Proc. Natl. Acad. Sci. USA 83:46314635. 8. Pizza, M. G., A. Covacci, A. Bartoloni, M. Perugini, L. Nencioni, M. T. De Magistris, A. Villa, D. Nucci, R. Manetti, M. Bugnoli, F. Giovannoni, R. Olivieri, J. T. Barbieri, H. Sato, and R. Rappuoli. 1989. Mutants of pertussis toxin suitable for vaccine development. Science 246:497-499. 9. Podda, A., L. Nencioni, M. G. De Magistris, A. Di Tommaso, P. Bossii, S. Nuti, P. Pileri, S. Peppoloni, M. Bugnoli, P. Ruggiero, I. Marsili, A. D'Errico, A. Tagliabue, and R. Rappuoli. 1990. Metabolic, humoral and cellular responses in adult volunteers immunized with the genetically inactivated pertussis toxin mutant PT-9K1129G. J. Exp. Med. 172:861-868. 10. Raubitschek, A. 1985. Epstein-Barr virus transformation, p. 454-455. In E. G. Engleman, S. K. H. Foung, J. Larrik, and A. Raubitschek (ed.), Human hybridomas and monoclonal antibodies. Plenum Press, London. 11. Sato, H., A. Ito, J. Chiba, and Y. Sato. 1984. Monoclonal antibody against pertussis toxin: effect on toxin activity and pertussis infections. Infect. Immun. 46:422-428. 12. Sato, H., and Y. Sato. 1990. Protective activities in mice of monoclonal antibodies against pertussis toxin. Infect. Immun. 58:3369-3374. 13. Sato, H., Y. Sato, A. Ito, and I. Ohishi. 1987. Effect of monoclonal antibody to pertussis toxin on toxin activity. Infect. Immun. 55:909-915. 14. Tamura, M., K. Nogimori, S. Murai, M. Yajima, K. Ito, T. Katada, M. Ui, and S. Ishii. 1982. Subunit structure of isletactivating protein, pertussis toxin, in conformity with the A-B model. Biochemistry 21:5516-5522.

Generation of human monoclonal antibodies that confer protection against pertussis toxin.

A panel of human monoclonal antibodies reactive with pertussis toxin has been generated by means of Epstein-Barr virus infection. One of these, the 3F...
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