Proc. Natl. Acad. Sci. USA Vol. 81, pp. 1809-1812, March 1984 Immunology

Shared idiotopes among monoclonal specific for A/PR/8/34 (HlNi) and X-31(H3N2) influenza viruses (shared idiotypes/influenza virus) THOMAS MORAN*, YUNG-NAN C. LIut, JEROME L. SCHULMAN*, AND CONSTANTIN A. BONA* *Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029; and tDepartment of Pathology, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115 Communicated by Edwin D. Kilbourne, December 2, 1983

ABSTRACT A monoclonal specific for the hem- MAbs. MAbs against PR8 virus were obtained from three agglutinin (HA) of influenza A X-31 (H3N2) virus (X-31) was independent fusions. In this study, we also used three MAbs obtained during a fusion of spleen cells from a BALB/c mouse obtained against the HA of influenza X-31 virus and one immunized with influenza A/PR/8/34 (HlNl) virus (PR8). MAb specific for the HA of B/Lee virus (see Table 1). Fu- This (Py2O6) shares crossreactive idio- sions were carried out with SP2/0 myeloma cells and spleen topes expressed on several monoclonal antibodies specific for lymphocytes from BALB/c mice immunized with influenza PR8 HA and X-31 HA as well as an individual idiotope shared viruses according to a previously described technique (15). with one monoclonal antibody specific for X-31 HA. The pres- MAbs specific for HA had antiviral hemagglutination-in- ence of shared idiotypy among antibodies of such diverse bind- hibiting and neutralizing activity and immunoprecipitated ing specificities may result from regulation by idiotype-specific HA of radiolabeled viral proteins. One antibody (Py2O3) im- T cells. In addition, the results suggest that, in contrast to the munoprecipitated the neuraminidase of radiolabeled PR8 vi- diversity of paratypes expressed, relatively few germ-line vari- rus and had neuraminidase-inhibiting activity in vitro. able region genes may be used in the antibody response to in- Determinations of Isotypes. Microtiter plates were coated fluenza A virus . with appropriate virus (50 ug/ml). After binding of MAbs, 3H-labeled goat anti-mouse subclass-specific antibodies Originally, idiotypes were considered to be phenotypic were added. After incubation at 40C overnight, the plates markers of variable-region genes and of the recep- were washed extensively, and radioactivity was measured in tors of B- and T-cell clones that cooperate in response to a a liquid scintillation counter. given antigen (1-3). However, shortly after idiotypes were Preparation of Anti-Idiotype Antibodies. Syngeneic anti- discovered, it was shown that antibodies reacting to the vari- idiotype antisera against MAbs Py202, Py206, Py207, XylO7, ous borne by the same antigen (4) or by different and XylO2 were obtained in BALB/c mice and homologous antigens (5-7) or even of unknown specificity (8, 9) share anti-idiotype antibodies against Py207 were obtained in A/J idiotopes. mice by a previously described technique (16). The homolo- Liu et al. (10) have shown that antibodies to various epi- gous anti-idiotype antisera were extensively adsorbed on topes on the hemagglutinin (HA) influenza A/PR/8/34 virus Sepharose 4B linked to UPC10 Ig2a to eliminate anti-allo- shared crossreactive idiotopes. This was surprising in light type activity. The anti-idiotype antibodies were purified by of several reports demonstrating great variability in the para- adsorption to Sepharose 4B coupled with the corresponding types to influenza virus HA as well as exquisite specificity in MAb and subsequent elution by 3 M MgCl2. the binding of anti-HA antibodies with closely related vari- Antigen Binding Activity. Antigen binding was determined ant viruses (11-13). in an ELISA by coating microtiter plates with purified PR8, During a fusion with spleen cells from a mouse immunized X-31, or B/Lee virus (50 gg/ml) for 1.5 hr. After extensive with influenza A/PR/8/34 (HlNi) virus (PR8), we found an washing with phosphate-buffered saline containing Tween antibody that bound to influenza A X-31 (H3N2) virus (X-31) and incubation with phosphate-buffered saline containing HA. In this communication, we present results that show 1% bovine serum albumin (P1/NaCl/albumin) for 1 hr, 10 ,ug that this particular monoclonal antibody (MAb) shares idio- of purified MAbs were added per ml and incubated over- topes expressed on MAbs obtained from three independent night. Alkaline phosphatase-labeled goat anti-mouse Ig anti- fusions specific for the PR8 virus HA and on MAbs specific bodies (New England Nuclear) were added. After 2 hr, the for X-31 virus HA. plates were washed extensively and p-nitrophenyl phosphate was added. After 1 hr at 370C, the reaction was stopped with 3 M NaOH, and the absorbance was read on a micro-ELISA MATERIALS AND METHODS minireader (Dynatech, Alexandria, VA) at 405 nm. Mice. Six- to 12-week-old BALB/c and A/J mice were ob- Assay for Idiotypes. Expression of idiotypes on MAbs tained from The Jackson Laboratory. were studied by hemagglutination (HA titer), hemagglutina- Viral Antigens. PR8 virus, X-31 virus, and influenza tion inhibition (HI), and ELISA. B/Lee/40 virus (B/Lee) were obtained from laboratory In hemagglutination and HI assays, MAbs were coupled to stocks and grown in the allantoic cavity of 10- to 11-day em- sheep erythrocytes (SRBC) by the CrCl3 method, and HA bryonated hens eggs. The viruses were collected and puri- and HI titers were determined by methods as described (9). fied by standard laboratory procedures (14). For immuniza- Titers were reported as 1/log2 of the highest dilution of anti- tion, mice were injected with 0.2 ml of allantoic seed virus bodies giving agglutination or inhibition of agglutination. diluted to contain 1000 HA units (15). Abbreviations: MAb, monoclonal antibody; HA, hemagglutinin; HI, The publication costs of this article were defrayed in part by page charge hemagglutination inhibition; IdX, cross-reactive idiotype; X-31, in- payment. This article must therefore be hereby marked "advertisement" fluenza A X-31 (H3N2) virus; PR8, influenza A/PR/8/34 virus; in accordance with 18 U.S.C. §1734 solely to indicate this fact. B/Lee, influenza B/Lee/40 virus; SRBC, sheep erythrocytes.

1809 Downloaded by guest on September 29, 2021 1810 Immunology: Moran et aL Proc. NatL Acad. Sci. USA 81 (1984)

Table 1. Antigen binding specificity and isotypes of MAbs used Table 2. Antigen binding specificity of various MAbs measured in this study by ELISA MAb Fusion Isotype Specificity* A45 of virus on microplate P20 1 y2b Hi MAb PR8 X-31 B/Lee P28 1 yl Hi Py202 1.23 0 0 PylO2 2 yi Hi Py202 3 y2b Py203 0.85 0.01 0.01 Hi Py206 0.03 1,25 0 Py203 3 y2a Ni Py206 3 y2b Py207 1.24 0.10 0.03 H3 Py210 1.28 0.02 Py207 3 y2b Hi 0 Py210 3 y2a Py211 0.83 0 0 Hi Py216 1.31 Py21l 3 y2b Hi 0 0 Py216 3 y2a XylO2 0.02 1.25 0.01 Hi BylO4 0.01 0 XylO2 4 y2a H3 1.18 XylO7 4 y1 H3 The binding of monoclonal antibodies to virus-coated plates was XylO8 4 y2a H3 measured by using alkaline phosphatase-labeled rabbit anti-mouse BylO4 5 y2a B Ig antibodies and measuring A at 405 nm. *H1, PR8 HA; N1, PR8 NA; H3, X-31 HA; B, B/Lee HA. fusions. Neither anti-Py202 nor anti-Py207 anti-idiotype anti- sera reacted with MAbs specific for X-31 (other than Py206). In ELISA, a very sensitive sandwich technique was used. In contrast, anti-Py206 antibodies recognized IdX expressed Briefly, microtiter plates were coated with purified MAbs (5 on MAb specific for PR8 as well as on two MAbs specific for pug/ml) overnight in coating buffer. After extensive washing X-31. and 1 hr of incubation with P1/NaCl/albumin, 10 ,ug ofaffini- Anti-idiotype antibodies against XyiO2 caused hemagglu- ty-purified anti-idiotype antibodies per ml were added to tination of SRBC coated with two MAbs specific for X-31 these plates and incubated overnight at 4°C. After extensive and SRBC coated with Py206. These results indicate that washing, alkaline phosphatase-labeled MAbs were added for Py2O6 shares idiotypes expressed on PR8-specific MAbs ob- 2 hr and washed, and substrate was added. The absorbance tained from three separate fusions and MAbs specific for X- was measured in the micro-ELISA reader at 405 nm. 31 virus. Analysis of Idiotopes of Py2O6 MAb. To further investigate RESULTS idiotopes expressed on Py206 MAb, we studied the direct binding of anti-idiotype antibodies to PR8- and X-31-specific Antigen Binding Specificity of Various MAbs Obtained MAb in an ELISA. Anti-Py206 antibodies and anti-XyiO2 from the Same Fusion. MAbs obtained from fusion 3 were antibodies bound very strongly to Py206, while anti-Py207 selected by their ability to bind PR8 virus. In this fusion, five bound weakly (Table 4). In addition, anti-XylO2 bound sig- MAbs were specific for PR8 HA, one (Py203) was specific nificantly to Py207-coated plates (Table 4). This latter result for PR8 neuraminidase, and Py206 bound to X-31 virus but was surprising in view of the absence of agglutination of not to PR8 virus (Table 1). In ELISA (Table 2), the binding SRBC coated with Py207 by anti-XylO2 (Table 3) but was of these MAbs was studied on microtiter plates coated with confirmed in subsequent ELISA. This suggests that at least PR8, X-31, or B/Lee virus. Py206 bound to X-31 virus-coat- some PR8-specific MAbs share an IdX with X-31-specific ed plates but not to PR8 virus-coated plates. In addition, this MAbs as determined by this assay. Interestingly, anti-Py206 antibody had high titers ofantiviral hemagglutination-inhibit- interacts strongly with idiotopes borne by Py207 and XylO2 ing and neutralizing activity against X-31 but not against PR8 and weakly to P20 and Py211. influenza virus (data not shown). Because ofthis unexpected These results were confirmed in the HI assay. In the first finding, we focused our studies on the idiotypy of Py2O6. system (Table 5), the ability of various MAb to inhibit the Specificity of Anti-Idiotype Antibodies. Syngeneic anti- agglutination of SRBC coated with P20 or Py211 by anti- idiotype antibodies were obtained by immunizing BALB/c Py207 was studied. In this system, an IdX shared by MAbs mice with one of three MAbs obtained from the same fusion from three independent fusions specific for PR8 virus and (Py202, Py206, and Py2O7) as well as MAbs specific for X-31 from Py206 was demonstrated. (XyiO2 and XyiO7). The specificities of these anti-idiotype In the second system (Table 6), Py206 was shown to bear antibodies were determined by hemagglutination assay. an IdX shared by XyiO2 and XylO7 as well as an IdX shared BALB/c anti-Py202 antibodies recognized crossreactive by XylO2 and XyiO8. Furthermore, Py206 also shared an idiotypes (IdXs) expressed on P20, PylO2, Py2O2 (antibodies "individual" idiotope expressed on XylO2. The data present- from three independent fusions), and Py206, but anti-Py2O2 ed in Table 7 shows that Py206 and XyiO2 inhibited the ag- antibodies did not recognize a shared idiotype on Py2O7 (a glutination of Py206-coated SRBC by anti-Py206 antibodies. MAb obtained from the same fusion as Py202 and Py2O6) The results taken collectively demonstrate that Py206, an (Table 3). X-31 HA-binding antibody, shares idiotopes both with anti- BALB/c anti-Py207 antibodies also recognized an IdX on bodies specific for HA of PR8 virus and antibodies specific antibodies specific for PR8 obtained from three independent for HA of X-31 virus. Table 3. Specificity of anti-idiotype antibodies determined by hemagglutination assays HA titer with coated SRBC, 102 units Anti-Id antibody P20 Py102 Py202 Py206 Py207 XyiO2 XylO7 XylO8 BALB/c anti-Py202 3 3 3 2 0 0 0 0 BALB/c anti-Py206 1 2 0 >8 1 >8 0 >8 BALB/c anti-Py207 2 2 1 4 >8 0 0 0 BALB/c anti-XyiO2 0 0 0 >8 0 >8 0 >8 Id, idiotype. Downloaded by guest on September 29, 2021 Immunology: Moran et aL Proc. Nat. Acad Sci. USA 81 (1984) 1811 Table 4. Direct binding of PR8 and X-31 HA-specific MAbs to Table 6. IdXs expressed on MAbs specific for X-31 HAs anti-idiotype antibodies in ELISA determined by HI assay A405 of idiotype-anti-idiotype system HI titer, log2 units Microtiter-plate Anti-Py207 Anti-XylO2 Anti-Py206 Anti-XylO7* Ant-Xy1O2* coating + Py207* + XylO2* + Py206* Inhibitor + XylO2-SRBC + XylO8-SRBC P20 0.02 0 0.11 P20 ND 0 Py202 0 0.03 0 Py206 7 >8 Py206 0.10 1.30 0.74 XylO2 7 >8 Py207 1.28 0.40 0.44 XylO7 6 3 Py211 0.12 0.07 0.12 XylO8 ND 6 XylO2 0.01 1.25 0.32 ND, not done. XylO7 0 0.01 0.07 *Anti-idiotype antibodies from BALB/c. *Alkaline phosphatase-labeled antibodies. occasionally after immunization of human subjects with A/New Jersey/8/76 (HlN1) virus (19). Moreover, DISCUSSION recently it was shown in a sensitive assay of antibody-depen- During a fusion of BALB/c spleen cells sensitized to PR8 dent cell-mediated cytotoxicity that children primed by pre- influenza virus, we immortalized a clone secreting MAbs vious natural infection with H3N2 viruses had increases in with specificity for the HA of X-31 influenza virus. All other concentrations of H3N2-specific antibody after immuniza- clones derived from this fusion were specific for PR8 and tion with HiN1 virus (20). showed no binding to X-31 HA. Finally, we considered the possibility that, in the response The binding of Py206 to various natural isolates of H3N2 to PR8, an idiotypic-specific helper T cell was induced. This variants determined by viral HI assay shows a remarkable T-cell helper might have favored the expansion of cells pro- specificity for only X-31 virus A/Aichi/2/68 (H3N2) virus ducing Py206 because it shares an idiotope with the other (from which X-31 derived its HA and neuraminidase genes) PR8-specific MAbs. Indeed, it has been shown that idiotype- and A/Eng/42/72 virus (data not shown). specific helper T cells are generated during a conventional To explain the derivation of this antibody with unusual immune response (21, 22). These cells can induce the expan- specificity, several possibilities must be considered. First, sioui of B-cell populations bearing the same idiotype in the during the clonal expansion after immunization, genetic absence of antigen (23, 24). In the influenza B virus system, events affecting the binding site could by chance have given Liu et al. (10) demonstrated in the secondary response of rise to a clone secreting an antibody specific for virus of a BALB/c mice the appearance of molecules sharing the IdX different subtype. This event must be extremely rare be- of anti-B/Lee antibodies but lacking antiviral specificity. cause, in a polyclonal antibody response, no neutralizing ac- Therefore, it is possible that the Py206 clone was stimulated tivity against X-31 virus is detected after PR8 virus immuni- in similar fashion. zation, despite the fact that the protein structures of the A detailed analysis of idiotopes expressed on Py206 MAb NH2-terminal subunits of HA, called HA1 subunits, show showed that this antibody shared (i) an IdX expressed by P20 35% homology (17). and Py211 and recognized by anti-Py207 antibodies; (ii) an Second, we cannot absolutely exclude the possibility that IdX expressed by XylO2 and XylO8 and recognized by anti- the animal was inadvertently primed with X-31 virus and XylO7 and anti-XylO2 antibodies, respectively; and (iii) an boosted with PR8 virus (although we have no evidence to additional "individual" idiotope shared by XylO2 as as- support this possibility). If this were the case, the activation sessed by the ability of only Py206 and XylO2 to inhibit the of an X-31 virus-specific clone could be related to the well- agglutination of Py206-coated SRBC by anti-Py206 (Table 7) known phenomenon of "original antigen sin." Accordingly, as well as by the ability of anti-Py206 and anti-XylO2 to bind among the clones expanded after primary immunization, Py206 and XylO2 MAb in an ELISA. there might have been some capable of interacting with PR8 We have considered several hypotheses to explain shared virus antigens, though programmed to secrete X-31 virus- idiotopes among Py206 and other MAbs. First, it should be specific antibody. Although original antigenic sin had not emphasized that by using syngeneic antisera in assays ade- been recognized previously in the responses to viruses as quate to detect IdXs, we observed IdXs among MAbs to dif- distantly related as H3N2 and HiN1 viruses (18), increased ferent determinants of PR8 virus HA obtained from three HI antibody titers to H2N2 and H3N2 viruses were observed independent fusions. Furthermore, IdXs were detected among MAbs specific for different virus-encoded polypep- Table 5. IdXs shared by MAbs obtained from three different tides-e.g., Py203, which is specific for PR8 virus neur- fusions specific for PR8 virus antigens and a MAb specific aminidase. Other MAbs specific for nucleoprotein and the for X-31 virus HA HA2 subunit of PR8 virus HA also have been shown to ex- HI titer, log2 units press the same IdXs (data not shown). These observations imply preferential activation of clones Anti-Py207Id* Anti-Py207Idt expressing IdXs by regulatory T cells. Such clones could be Inhibitor + P20-SRBC + Py211-SRBC derived from common ancestral variable region genes. These P20 6 4 PylO2 5 2 Table 7. Individual idiotypic specificity shared by two MAbs Py202 3 >8 obtained from different fusions specific for X-31 HAs Py203 ND 6 HI titer, log2 units Py206 >8 >8 Py211 ND >8 MAb Anti-Py206 + Py206-SRBC B118 0 ND P20 0 ND, not done. Py206 >8 *Anti-idiotype antibodies from BALB/c. XylO2 >8 tAnti-idiotype antibodies from A/J mice. XylO7 0 Downloaded by guest on September 29, 2021 1812 Immunology: Moran et aL Proc. NatL. Acad. Sci. USA 81 (1984)

genes coding for antibodies specific for different influenza 7. Hiernaux, J. & Bona, C. A. (1982) Proc. Natl. Acad. Sci. USA viruses may have conserved common idiotypes while diverg- 79, 1616-1620. ing in their antigen-binding specificity. 8. Eichmann, K., Coutinho, A. & Melchers, F. (1977) J. Exp. Second, antibody responses to a spectrum of influenza vi- Med. 146, 1436-1448. rus antigenic determinants may use 9. Bona, C., Mond, J. J., Stein, K. E., Howe, S., Lieberman, R. a few germ-line genes. & Paul, W. E. (1979) J. Immunol. 123, 1484-1490. Subsequent to somatic rearrangements and/or mutational 10. Liu, Y.-n. C., Bona, C. A. & Schulman, J. L. (1981) J. Exp. events and clonal expansion induced by antigen, antibodies Med. 159, 1525-1538. are produced that conserve segments of DNA encoding an 11. Gerhard, W., Croce, C. M. & Koprowski, H. (1978) Proc. IdX but producing antibodies with different specificities. In Natl. Acad. Sci. USA 75, 1510-1514. the CRI' Ars system (crossreactive idiotypic-positive ars- 12. Cancro, M. P., Wylie, D. E., Gerhard, W. & Klinman, N. R. anilic acid system), it was clearly shown that antibodies en- (1979) Proc. Natl. Acad. Sci. USA 76, 6577-6581. coded by germ-line genes in association with joining region 13. Staudt, L. M. & Gerhard, W. (1983) J. Exp. Med. 157, 687- JH2 genes are Ars', whereas CRI+ Ars- antibodies use the 704. same germ-line gene in association with JH4 (25). It is possi- 14. Palese, P. & Schulman, J. L. (1974) Virology 57, 227-237. 15. Lubeck, M. D., Schulman, J. L. & Palese, P. (1980) Virology ble that similar mechanisms were involved in the generation 102, 458-462. of Py206. 16. Bona, C. & Paul, W. E. (1979) J. Exp. Med. 149, 532-600. In conclusion, we have studied the idiotypes of various 17. Palese, P. & Young, J. F. (1982) Science 215, 1468-1474. influenza virus-specific MAbs and demonstrated a sharing of 18. Webster, R. G. (1966) J. Immunol. 97, 177-183. idiotypes among antibodies with specificities for different vi- 19. Noble, G. R., Kaye, H. S., Kendal, A. P. & Dowdle, W. R. ruses. (1977) J. Infect. Dis. 136, S686-S692. 20. Hashimoto, G., Wright, P. F. & Karzon, D. T. (1983) J. In- This work was supported by Grants A114053 and A118316 from fect. Dis. 148, 785-794. the U.S. Public Health Service. 21. Cerny, J. & Caulfield, M. J. (1981) J. Immunol. 126, 2262- 2266. 1. Kunkel, H. G., Mannick, M. & Williams, R. C. (1963) Science 22. Julius, M. H., Augustin, A. A. & Cosenza, H. (1977) Nature 140, 1218-1221. (London) 265, 251-253. 2. Binz, H. & Wigzell, H. (1975) J. Exp. Med. 142, 197-211. 23. Bona, C. (1979) in Regulation of Lymphocyte Function by 3. Eichmann, J. & Rajewsky, K. (1975) Eur. J. Immunol. 5, 661- Anti-Idiotype Antibody in the Molecular Basis ofImmune Cell 666. Function, ed. Kaplan, J. (Gordon & Breach, New York), pp. 4. Cazenave, P. A. (1973) FEBS Lett. 31, 348-354. 161-180. 5. Oudin, J. & Cazenave, P. A. (1971) Proc. Natl. Acad. Sci. 24. Eichmann, K., Falk, I. & Rajewsky, K. (1978) Eur. J. Immu- USA 68, 2610-2620. nol. 8, 853-857. 6. Ju, S.-T., Benacerraf, B. & Dorf, M. E. (1980) J. Exp. Med. 25. Margolies, M. N., Wysocki, L. J. & Sato, V. L. (1983) J. Im- 152, 170-182. munol. 130, 515-517. Downloaded by guest on September 29, 2021