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Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+. Catalysis was associated with BV 04-01 IgG, Fab, and single-chain-antibody (SCA) proteins. Cleavage of both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of A7C7ATATAGCGCGT2, as well as a preference for cleaving within CG-rich regions of dsDNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA, and two SCA mutants were used to model the catalytically active antibody site using the previously resolved X-ray structure of BV 04-01. The resulting model suggested that the target phosphodiester bond is activated by induction of conformational strain. In addition, the antibody-DNA complex contained a Mg2+ coordination site composed of the L32Tyr and L27dHis side chains and a DNA 3'-phosphodiester group. Induction of strain along with the metal coordination could be part of the mechanism by which this antibody catalyzes DNA hydrolysis. Sequence data for BV 04-01 V(H) and V(L) genes suggested that the proposed catalytic-antibody active site was germline-encoded. This observation suggests that catalytic activity might represent an important-rarely examined-function for some antibody molecules.

Individual antigenic determinants on antibodies were originally described by Kunkel et al. (1). Subsequently, the determinants, termed "idiotypes" by Oudin (2), have been described in antibody populations made in response to bacterial antigens (3) and to haptens (4). The antiidiotypic antisera are usually made in homologous or even heterologous species. Absorption of the antiidiotypic antisera with preinoculation serum or IgG from the individual whose idiotype is being studied might not remove antibodies specific for constant areas of variable (V) 1 regions of a particular V-region subclass represented in the antibody (idiotype) population. Certainly, allotype matching of donor and recipient restricts one to matching for only the presently known allotypes. Idiotypic probes of V-region genetics might be more useful if a technique could be used that would elicit only antibodies in the antiidiotypic antiserum specific for hypervariable regions in the idiotypic population.Evidence is presented here that determinants characteristic of idiotypes can induce synthesis of "autoantiidiotypic antibodies" within the same individual. This system might serve as a tool for studying hypervariable-region genetics and provide a different approach to questions of autoimmune processes. Materials and MethodsAnimals.--Outbred New Zealand White rabbits were used.Haptens.--The hapten p-aminophenyl-N-trimethylammonium chloride (R4N) was obtained from Dr. V. Peter Kreiter, Glenwood, N.Y. Further purification was done by recrystallizing R4N from methanol. N,N-dimethyl-p-phenylenediamine dihydrochloride (R3N) was obtained from the Eastman Kodak Co., Rochester, N.Y. and was purified further by recrystallization from ethanol, p-nitrobenzoic acid (PNBA) was obtained from Matheson Coleman and Bell, East Rutherford, N.J. and was recrystallized from ethanol.Antigen.--The hapten R4N was coupled to keyhole limpet hemocyanin (KLH) by diazotization at pH 9-9.5 using a weight ratio of 40 mg/g KLH. The KLH-R4N conjugate was

The antibody response of a single outbred rabbit was studied throughout three rounds of injections with Micrococcus lysodeikticus vaccine over a 31-mo period. The first-round response was characterized by a vigorous anti- micrococcus response and a strong anti-IgG rheumatoid factor response. The second-round response consisted of a triad of interacting molecules: anti- micrococcal antibodies, autoanti-idiotypic antibodies specific for distinct clonotypes of the first-round anti-micrococcal antibodies, and Fc-specific anti-IgG rheumatoid factor. The interacting triple complex was detected because of the formation of an immune complex that became insoluble upon dilution of the serum. Complex formation was inhibited in the presence of saccharide compounds known to be major immunodominant determinants of the micrococcal cell-wall carbohydrate polymer. The same saccharides did not affect the reaction of rheumatoid factor with IgG. Direct-binding radioimmunoassays ruled out mediation of the dilution-precipitation reaction by soluble micrococcal antigens. Specific absorption of rheumatoid factor inhibited the dilution-precipitation reaction. Auto-anti-idiotypic antibodies were specifically purified from second-round sera, directly confirming the presence of these antibodies. Suppressive effects of auto-anti-idiotypic antibodies on distinct antibody clonotypes were shown by gel isoelectric focusing of first-, second-, and third-round sera. Clonotypes expressed in the first round of immunizations were reduced in quantity or absent when auto-anti-idiotypic antibodies were detectable. Greatly enhanced levels or initial synthesis of new clonotypes of anti-micrococcal antibodies were detected during the period of auto-anti-idiotype synthesis. The third-round sera, devoid of detectable auto-anti-idiotype, contained clonotypes characteristic of both first- and second-round antisera. Thus, auto-anti- idiotypic-mediated suppression appeared to be reversible. The data are interpreted as lending strong support for concepts of autoregulation of immune processes in normal outbred animals via an idiotypic network.

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Intraveneous hyperimmunization of selectivity bred rabbits with streptococcal group A-variant vaccines elicits antibody responses of restricted heterogeneity at high antibody levels. All antisera contain two functionally distinct antibody populations, which can be isolated in single-band purity upon analytical isoelectric focusing. Typical examples of these two kinds of single-band antibodies were investigated in great detail for several parameters by a variety of methods. 85--99% of the streptococcal group A-variant polysaccharide (Av-CHO)-specific antibody in the antisera does not precipitate the isolated 5,000 daltons poly-L-rhamnose antigen, neither agglutinates nor lyses in the presence of complement Av-CHO-coated sheep erythrocytes (SRBC), binds the radio-labeled Av-CHO with an association constant in the ragne of 10(5)--10(6) M-1, and is of terminal specificity (nonreducing end) for the linear Av-CHO. In contrast, the minor fraction of Av-CHO-specific antibody (1--15%) does precipitate the linear Av-CHO, both agglutinates and lyses Av-CHO-coated SRBC in the presence of complement, has an affinity range of 10(8)--10(9) M-1, and is of internal specificity for the Av-CHO. The antigenic determinants of the Av-CHO for the antibodies are nonoverlapping, only one Fab of the low affinity antibody can be bound whereas four Fab of the high affinity antibody are accommodated. Hence, the determinant specificity explains the functional differences observed, for there is no indication of subclass differences. A mechanistic model of the A-variant carbohydrate presentation on the vaccine appears to account best for the unbalanced levels of low and high affinity antibody.

We have previously demonstrated molecular mimicry between the S peplomer protein of mouse hepatitis virus (MHV) and Fc gamma R (Fc gamma R). A monoclonal antibody (MAb) to mouse Fc gamma R (2.4G2 anti-Fc gamma R MAb), purified rabbit immunoglobulin, but not their F(ab')2 fragments, as well as mouse and rat IgG, immunoprecipitated (1) recombinant S peplomer protein expressed by a vaccinia virus recombinant in human, rabbit, and mouse cells, and (2) natural S peplomer protein from cells infected with several strains of MHV and MHV escaped mutants. We report here results of studies documenting molecular mimicry between Fc gamma R and S peplomer protein of viruses representing three distinct antigenic subgroups of the Coronaviridae. We have shown a molecular mimicry between the S peplomer protein of bovine corona virus (BCV) and Fc gamma R. The 2.4G2 anti-Fc gamma R MAb, rabbit IgG, but not its F(ab')2 fragments, as well as homologous bovine serum, free of anti-BCV antibodies, immunoprecipitated S peplomer protein of BCV (Mebus strain). In contrast, we did not find molecular mimicry between S peplomer protein of human corona virus (HCV-OC43) and Fc gamma R. Although the OC43 virus belongs to the same antigenic group as MHV and BCV, MAb specific for human Fc gamma RI or Fc gamma RII and purified human IgG1, IgG2, and IgG3 myeloma proteins did not immunoprecipitate the S peplomer protein from HCV-OC43-infected RD cells. In addition, we did demonstrate molecular mimicry between the S peplomer protein of porcine transmissible gastroenteritis virus (TGEV) and Fc gamma R. TGEV belongs to the second antigenic subgroup of coronaviridae.(ABSTRACT TRUNCATED AT 250 WORDS)

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