Effects of various adjuvants and a viral infection on the antibody specificity toward native or cryptic epitopes of a protein antigen.

An immunization protocol that induces antibodies (Abs) directed to cryptic epitopes of a protein antigen (Ag) reduces the efficacy of vaccines that ideally should induce Abs against native epitopes. We have shown earlier that viral infections concomitant with immunization against a protein tend to shift the Ab specificity toward cryptic epitopes and tend to induce the production of autoantibodies (autoAbs). Here, we show the effects of three adjuvants on the Ab specificity in the absence or presence of a viral infection (lactate dehydrogenase-elevating virus or LDV), with human growth hormone (hGH) being, as before, the protein Ag. Pathogen-free CBA/Ht and BALB/c mice were immunized with hGH in the presence of complete Freund's adjuvant (CFA), monophosphoryl lipid A (MPL) or alum, with the animals being either infected with LDV or not infected with LDV. Conventional and competition enzyme-linked immunosorbent assays (ELISAs) indicated that in noninfected mice, CFA induced higher titres of anti-hGH Ab than did MPL or alum, with the Ab being almost totally directed to cryptic hGH epitopes. Strikingly, CFA plus LDV infection in CBA/Ht mice shifted the specificity of the anti-hGH Ab toward native epitopes, whereas the virus decreased the Ab titre when MPL or alum was used. Our Western blot results showed that 70% of mice immunized with hGH in the presence of any adjuvant produced autoAbs against a variety of tissue Ags. The amount of autoAb and the concentration of Ab to hGH cryptic epitopes did correlate, suggesting a relationship between both kinds of Ab. Significant differences were observed in the various effects of adjuvants and the viral infection between the two mouse strains used in this work.

Relative localization of the prolactin receptor binding sites for lactogenic hormones.

A monoclonal antibody termed MAb R7B4, directed to an epitope present in prolactin receptors (PRLRs), was used as a tool to map the receptor binding sites for human growth hormone (hGH), ovine prolactin (oPRL) and human placental lactogen (hPL). Although the three hormones completely inhibited the binding of each other to Nb2 cells or rat liver receptors, MAb R7B4 behaviour was different depending on the hormone tested and the receptor source. According to the MAb effects, PRLR from Nb2 cells would locate both hGH and oPRL close to R7B4 epitope, whereas hPL would bind far from the MAb binding site. On the other hand, PRLR from rat liver should bind hGH close to the R7B4 epitope but oPRL and hPL would be recognized by a separate region of the same receptor. Thus, results presented in this paper suggest that PRLR binding sites for hGH, oPRL and hPL do not exactly overlap in spite of full competition between ligands.

Ovine placental lactogen and human growth hormone bind to different regions of the same receptors.

Anti-human growth hormone (hGH) polyclonal and monoclonal antibodies (MAb) failed to recognize ovine placental lactogen (oPL), indicating that the antigenic topographies of both hormones are different. Binding assays showed that oPL completely inhibited hGH binding to lactogenic receptors from Nb2-cells and to somatogenic receptors from rabbit or sheep liver; in contrast, oPL only bound to a subpopulation of rat liver receptors. Zinc ion increased hGH and oPL binding to Nb2-cell receptors and slightly inhibited both hormones' recognition by somatogenic receptors. However, ZnCl(2) increased hGH binding to rat liver microsomes but prevented that of oPL. Furthermore, MAb R7B4, recognizing lactogenic as well as somatogenic receptors, entirely blocked hGH binding to the various receptor systems but not affected oPL binding. Therefore, results presented in this paper suggest that oPL and hGH bind to different regions of the same receptors.

A monoclonal antibody recognizing an epitope shared by receptors for growth hormone, prolactin, interleukin 2 and interleukin 6.

Monoclonal antibody (MAb) termed R7B4 was generated throughout the idiotypic-anti-idiotypic network from mice immunized with human and bovine growth hormones (GH). The Ab was selected on the basis that it did not recognize human GH (hGH) neither insolubilized nor in solution but inhibited 125I-hGH binding to receptors from rat and rabbit liver and from Nb2-cell membranes. Since it inhibited Nb2-cell mitogenesis stimulated by hGH, prolactins or placental lactogens, MAb R7B4 behaved as an antagonist of lactogenic hormones. Furthermore, the Ab impaired proliferative activity of interleukin 2 (IL-2) on Nb2 cells as well as growth of 7TD1 cells, an interleukin 6 (IL-6) dependent hybridoma not expressing GH receptors. Biotin-labeled MAb R7B4 specifically bound to rat liver microsomes, and the Ab was able to recognize Nb2 and 7TD1-cell membranes as shown by flow cytometry experiments. However, MAb binding was not hampered by hGH, indicating that the Ab did not mimic GH binding site to receptors. Immunoblot assays indicated that rat and rabbit liver as well as Nb2-cells membrane antigens recognized by MAb R7B4 were similar to those revealed by a MAb directed to prolactin receptors. In addition, MAb R7B4 was able to detect two bands probably corresponding to the somatogenic receptor in rabbit liver microsomes as well as three different proteins in 7TD1-cells showing molecular weights similar to those of the IL-6 receptor complex. Results suggest that MAb R7B4 is directed to an epitope shared by receptors for lactogenic and somatogenic hormones, IL-2 and IL-6. To our knowledge, these data are the first experimental evidence of the existence of structural similarity between some of the receptors grouped in the cytokine receptor superfamily.