Molecular cloning and expression of the Fabs of human autoantibodies in Escherichia coli. Determination of the heavy or light chain contribution to the anti-DNA/-cardiolipin activity of the Fab

The Fabs of three human autoantibodies (B3/33H11, anti-DNA; UK4, anti-phospholipid) and six related hybrids have been cloned, expressed in Escherichia coli, and purified to homogeneity. SDS-polyacrylamide gel electrophoresis and Western blot analysis of the recombinant Fab demonstrated the purified Fab to be of correct size and in assembled form. Protein expression levels of up to 5-9 mg per liter of culture were achievable. A sensitive and reliable comparative anti-DNA enzyme-linked immunosorbent assay, involving a defined biotinylated 35-mer oligonucleotide in its single- or double-stranded form, is also described. Crithidia assay and anti-DNA or anti-cardiolipin antibody enzyme-linked immunosorbent assay analyses demonstrated convincing binding of the recombinant Fab proteins to DNA/cardiolipin, confirming the expression of functional molecule. The comparative DNA/cardiolipin binding analyses of the nine Fabs revealed that the anti-DNA (light, B3/33H11) or anti-cardiolipin (heavy, UK4) activity lies predominantly on one of the two chains. However, a compatible partner chain is necessary for optimum antigen binding activity of the antibody.     

Quantitative aspects of lupus anti-DNA autoantibody specificity

In this study we have attempted to define the cross-reactive potential of SLE anti-DNA antibodies (in 19 representative sera and plasmas) in both the solution phase and the solid phase. We used the Farr and RBC-CF solution phase assays to measure quantitatively the ability of a variety of negatively charged structurally unrelated molecules to inhibit antibody binding to both native DNA (nDNA) and denatured DNA (dDNA). The inhibitors used were of two types: 1) phospholipids (cardiolipin, phosphatidyl glycerol, and phosphatidic acid) and 2) repeating negatively charged molecules (poly-glutamic acid, heparin sulfate, and chondroitin sulfate). We found in both assays that the phospholipids could inhibit antibody binding to nDNA and dDNA, but a large excess (about 1500-fold) of these molecules was needed relative to DNA to achieve equivalent levels of inhibition. The repeating negatively charged molecules did not inhibit DNA binding at equivalent molar levels as the phospholipids; generally, at least a 10,000-fold excess was needed relative to the nucleic acids to achieve any appreciable inhibition. Results of a dDNA binding-inhibition solid-phase ELISA for cross-reactivity of the anti-DNA antibodies gave quite similar results. Finally, we found that eight of the SLE samples did have anti-cardiolipin antibodies, as demonstrated in a cardiolipin-based ELISA. These results suggest that previous reports describing an apparent cross-reactivity of anti-DNA antibodies may not represent physiologically relevant interactions between anti-DNA antibodies and non-nucleic acid antigens.     

The serology and immunochemistry of HIV-induced platelet-bound immunoglobulin

A study was carried out on the presence of platelet-bound immunoglobulins, platelet-bound complement and serum immunoglobulin reactive with platelets in the blood of persons infected with HIV and those at risk of HIV infection. Platelet-bound immunoglobulins, predominantly IgG and IgM, but not complement, were demonstrated by immunofluorescence in 16 out of 16 patients with AIDS, in 5 out of 7 with AIDS-related complex/persistent generalized lymphadenopathy and in 7 out of 10 apparently healthy sexually active homosexual men, of whom 2 were anti-HIV1 seropositive. There was no correlation between the presence of platelet-bound immunoglobulins and either the platelet count or the level of circulating immune complexes. The specificity of the platelet-bound immunoglobulins and platelet-reactive immunoglobulins in the corresponding sera was studied. Platelet-bound immunoglobulins were eluted and then investigated for cross-reactivity with HIV. Both sera and eluates were tested for reactivity with cardiolipin and reactivity with the major target antigen in classical autoimmune thrombocytopenia, the GP IIb/IIIa complex. Of 17 eluates containing platelet-reactive immunoglobulins, 5 reacted with HIV-determinants but 2 out of 5 eluates that did not contain platelet-reactive immunoglobulins also reacted. Although anti-cardiolipin antibodies were detected in all sera, none of the 17 eluates reacted with cardiolipin. Moreover, sera and eluates, reactive with normal platelets, did not react with type-1-Glanzmann disease platelets. This indicates that the antibodies are directed against the glycoprotein IIb/IIIa complex of platelets. This could not be confirmed by immunoprecipitation or by immunoblotting, however. We conclude that the presence of platelet-bound immunoglobulins is common in HIV-infection but may also occur in persons at risk and that the nature of the auto-antibodies is not different from that of the auto-antibodies observed in classical ITP.     

A DNA antigen that reacts with antisera to cardiolipin

DNA can replace the cardiolipin hapten in an antigen suspension that precipitates anti-cardiolipin antibody. Structural similarities between cardiolipin and DNA may explain the immunochemical cross reaction between the nucleic acid and the phospholipid molecule.     

Reaction patterns of monoclonal antibodies to DNA

Starting with spleen cells from MRL/lpr, NZB/W, and graft-vs-host-diseased mice, we prepared a total of 57 hybridomas that produce antibodies to DNA. Using various approaches, we studied the avidity of these monoclonals in relation to their behavior in four anti-DNA assays. From the results obtained, we postulate that on the basis of anti-DNA avidity the anti-DNA ELISA, the polyethylene glycol assay, the indirect immunofluorescence test on Crithidia luciliae, and the Farr assay (in this order) detect a decreasing amount of anti-dsDNA, the Farr assay being strictly selective for high avidity anti-dsDNA. mAb selected by the anti-DNA ELISA generally were of a low avidity toward DNA. Using cardiolipin and dextran sulfate, a polyanion that bears a resemblance in charge to DNA, we studied the cross-reactivity of the monoclonals. A total of 6 of the 57 monoclonals were found to cross-react with cardiolipin, and 26 with dextran sulfate. We observed an inverse relationship between anti-DNA avidity and cross-reactivity: the lower the avidity of the antibody, the more cross-reactive it is. Based on these findings, we postulate that it is at least questionable whether low avidity, cross-reactive (monoclonal) anti-DNA is representative for the anti-DNA found in patients with SLE.     

Hybridoma anti-DNA autoantibodies from patients with rheumatoid arthritis and systemic lupus erythematosus demonstrate similar nucleic acid binding characteristics

Hybridoma anti-DNA antibodies have been generated from the fusion of the GM 4672 lymphoblastoid line with peripheral blood lymphocytes from four normal subjects, nine patients with rheumatoid arthritis (RA), and 13 patients with systemic lupus erythematosus (SLE). A total of 441 hybridoma clones were obtained, of which 37 secreted anti-DNA autoantibodies. The nucleic acid binding characteristics of the anti-DNA antibodies produced by two hybridomas from normal subjects, nine hybridomas from RA patients, and 18 hybridomas from SLE patients are reported. The hybridoma anti-DNA antibodies from all three groups showed similar antigen-binding characteristics for denatured DNA (dDNA), native DNA (nDNA), poly(I), poly(dT), and cardiolipin, by both direct binding and competitive binding analyses. One difference noted between normal-derived anti-DNA antibodies and autoimmune-derived antibodies was the inability of the former to react with z-DNA. However, this requires further substantiation with larger numbers of normal-derived clones. The broad overlap of reactivity to nucleic acid antigens among individual anti-DNA autoantibodies found in two clinically different autoimmune diseases, namely RA and SLE, suggests that the pathogenicity of anti-DNA autoantibodies may bear no relationship to their nucleic acid antigen-binding characteristics.     

Influenza PR8 HA-specific Fab fragments produced by phage display methods

Anti-influenza hemagglutinin (HA) Fabs were isolated from a phage display library using purified HA of influenza virus A/Puerto Rico/8/34 (PR8; H1N1) as an antigen. Four Fab clones displaying a 25-50-fold higher binding signal to PR8 HA than the control were selected for further analysis and comparison with anti-PR8 monoclonal antibody (mAb). All four Fabs and mAb recognized the PR8 HA under non-reducing conditions but rarely bound to reduced PR8 HA. Inhibition of influenza virus infection on MDCK cells was observed with Fab1 and mAb in a dose-dependent manner while Fab3 and 4 exhibited only a partial inhibitory effect. Moreover, Fab1 clone and mAb exhibited cross-reactivity with the A/Peking/262/95 (A/Peking; H1N1) strain. The inhibitory effects of mAb on both influenza strains were more potent than Fab1, which is likely attributed to its higher affinity for the antigen. SPR analyses, in fact, revealed that Fab1 and mAb have K_D of 1.5 × 10⁻⁸ and 3.2 × 10⁻⁹ M, respectively. These results strongly suggest that phage library-derived Fabs can be readily prepared and that such HA-specific Fabs with inhibitory action on influenza infection may be used to treat influenza patients.     

Alpha-actinin is a cross-reactive renal target for pathogenic anti-DNA antibodies

Anti-DNA Abs commonly found in patients with systemic lupus erythematosus are thought to play an important pathogenic role in lupus nephritis. Anti-DNA Abs may contribute to renal disease by cross-reactivity with renal Ags, the identity of which remain elusive. To identify a target Ag for pathogenic anti-DNA Abs, we performed Western blotting and immunoprecipitations of mesangial cell lysates from the lupus-prone MRL-lpr/lpr mouse and a nonautoimmune BALB/c mouse with the pathogenic anti-DNA Ab R4A. We found that R4A (but not a nonpathogenic Ab mutant of R4A) binds to and immunoprecipitates a 100-kDa protein expressed on the cell surface and in lysates of MRL-lpr/lpr mesangial cells. DNase treatment of the lysate and of the R4A Ab did not effect binding, indicating that the binding of R4A to the 100-kDa protein was direct and not mediated by an antigenic bridge containing DNA. Binding was greatly diminished in BALB/c lysates, suggesting that Ag expression or availability at the level of the target organ may be a factor in determining susceptibility to lupus nephritis. Following identification of this 100-kDa protein as nonmuscle alpha-actinin, binding of R4A to alpha-actinin was confirmed by Western blot, ELISA, inhibition studies, and immunofluorescence. High titers of anti-alpha-actinin Abs were present in sera and kidney eluates of lupus mice with active nephritis. These results indicate that the nephritogenicity of some anti-DNA Abs may be mediated via cross-reactivity with alpha-actinin. Furthermore, variations in target Ag display between individuals may underlie differential susceptibility to anti-DNA Ab-induced renal disease.     

Specific and shared idiotypes found on hybridoma anti-DNA autoantibodies derived from rheumatoid arthritis and systemic lupus erythematosus patients

The idiotype determinants found on hybridoma anti-DNA autoantibodies produced from the fusion of peripheral blood lymphocytes from 13 systemic lupus erythematosus (SLE) and five rheumatoid arthritis (RA) patients with the GM 4672 human lymphoblastoid line were analyzed. A total of 47 SLE and 21 RA hybridomas were studied, of which 26 SLE and 10 RA produced anti-DNA autoantibodies. Rabbit antisera, raised to six of the SLE hybridoma anti-DNA IgM antibodies, were rendered idiotype specific by multiple absorptions on human IgM and IgG immunoabsorbent columns. In direct binding radioimmunoassays, all six anti-idiotype antisera reacted specifically with the anti-DNA antibody used as immunogen. In competition studies, five anti-idiotype antisera were able to inhibit the binding of their homologous idiotype to DNA-coated tubes. In addition, DNA and polynucleotides inhibited the binding of the five idiotypes to anti-idiotype-coated tubes, suggesting that these anti-idiotypes react with idiotype determinants located within the antigen-combining sites of the anti-DNA antibody molecules. Shared idiotypes were detected among the 68 hybridoma antibodies by direct binding studies on anti-idiotype-coated tubes. Our results revealed that 58% (21/36) of the anti-DNA antibodies and 16% (5/32) of the non-DNA-binding antibodies reacted with at least one anti-idiotype serum. Five anti-idiotype antisera reacted only with hybridoma anti-DNA antibodies from SLE patients. The other anti-idiotype antiserum reacted with both SLE- and RA-derived hybridoma anti-DNA and non-DNA-binding antibodies. These studies indicate that some anti-idiotype antisera may detect specific idiotypes found only on SLE-derived anti-DNA auto-antibodies, whereas other antisera detect shared idiotypes found on both RA and SLE DNA-binding and non-DNA-binding antibodies.     

Base specificity and idiotypy of anti-DNA autoantibodies reactive with synthetic nucleic acids

Synthetic nucleic acid reactivities and the distribution of idiotypes associated with poly(dA) and poly(dT) specificities were evaluated among both monoclonal and polyclonal anti-DNA antibodies from autoimmune New Zealand mice. Ten monoclonal anti-DNA antibodies (IgG2a or IgG2b), derived from NZB/NZW mice and reactive with natural DNA (duplex and/or heat-denatured), were found to collectively exhibit a diverse binding pattern with six deoxyribohomopolymers. Several monoclonal antibodies displayed reactivity with poly(dT) comparable to that with natural DNA. Serologic studies indicated that polyclonal anti-DNA autoantibodies from NZW/NZW mice and both parental strains also cross-reacted with various homopolymers and bound preferentially with those containing pyrimidines, particularly poly(dT), relative to purines. Detailed binding analyses with two poly(dT)-reactive monoclonal antibodies demonstrated that stable DNA/anti-DNA complexes were formed with synthetic oligomers containing six to 10 nucleotides; binding to such antigens was relatively insensitive to ionic strength and inversely dependent on temperature. Both antibodies exhibited preferential binding (greater than or equal to 10-fold) with poly(dT) relative to poly(dU), suggesting the importance of the C5-methyl group and/or helical conformation in pyrimidine base recognition. Idiotypes on poly(dA)-specific and poly(dT)-specific monoclonal antibodies were found to be reciprocally distinct, localized at or near active site residues, and expressed at low levels (less than 10 to 130 ng/ml) in anti-DNA sera from all three New Zealand strains. These findings suggest that: nucleotide base determinants are significantly involved in DNA/anti-DNA interactions; poly(dT) represents a major cross-reactive synthetic antigen; and idiotype expression among lupus autoantibodies which recognize such determinants may be diverse.     

Murine monoclonal anti-DNA autoantibodies bind to endogenous bacteria

Several bacterial species (including Streptococcus faecalis, Bacillus cereus, Staphylococcus aureus, and Escherichia coli) were tested for their ability to react with monoclonal anti-DNA antibodies that were derived from MRL-lpr/lpr mice. S. faecalis reacted with 8/15 of such antibodies. The binding was unaffected by DNase, but it was competitively inhibited by DNA. F(ab')2 fragments of the monoclonal antibodies reacted with the bacteria, but Fc fragments did not. Phospholipids extracted from the bacterial cells were able to bind to three representative anti-DNA antibodies that also bound to whole bacteria. The results suggest that bacterial phospholipids might provide an immunogenic stimulus for the production of antibodies that cross-react with DNA. We propose that some anti-DNA auto-antibodies and anti-bacterial antibodies evolve from a restricted group of antibodies with high avidity for the phosphodiester groups that occur in DNA and bacterial cells walls.     

Histone H1; A neuronal protein that binds bacterial lipopolysaccharide

Bacterial lipopolysaccharide (LPS) is a potent inflammogen following systemic infection. Macrophages express a number of surface molecules including CD14, CD18 and the scavenger receptor that are capable of recognizing and binding LPS. Injection of the CNS with LPS produces an atypical inflammatory response including a delay in the recruitment of macrophages to the brain parenchyma. We have shown using a ligand blot overlay approach, that LPS is capable of binding to histone H1 present in brain homogenate. The ability of LPS to bind to H1 has only been previously shown for monocytes. Subsequent immunohistochemistry revealed that the anti-H1 antibody, ANA-108, stained neuronal cell bodies and was located in the membrane, possibly at the cell surface. Further experiments revealed that the H1 antigen recognized by the ANA-108 antibody was not a histone wholly restricted to the nucleus but may represent a novel CNS form of the protein. This observation has implications for the autoimmune disease systemic lupus erythematosus (SLE) due to the presence of auto-antibodies, particularly against DNA and nuclear proteins, in serum. The formation of immune complexes in various organs leads to severe dysfunction. Anti-histone antibodies are typical of the auto-antibodies found in SLE serum and the presence of the H1 antigen on the surface of neurons could provide an insight into biology underlying the neurological problems associated with SLE.     

Crystal structures of the 64M-2 and 64M-3 antibody Fabs complexed with DNA (6-4) photoproducts.

Crystal structures of the 64M-2 antibody Fab fragment complexed with DNA photoproducts of dT(6-4)T and dTT(6-4)TT, and of the 64M-3 Fab fragment complexed with dT(6-4)T were determined. The 5'-thymine base of the bound dT(6-4)T ligand is in a half-chair conformation, and its base plane is nearly perpendicular to the planar 3'-pyrimidone base. The 64M-2 and 64M-3 Fabs have a common structure suitable for accommodating the dT(6-4)T ligand. In each of the antigen binding sites of the 64M-2 and 64M-3 Fabs, basic residues of His 35H and Arg 95H are located at the bottom of the binding pocket, and are hydrogen-bonded to the base moieties of dT(6-4)T. Two water molecules are involved in the interactions that intervene between the base moieties and the binding site. Aromatic residues of Trp 33H and Tyr 100iH form a side-wall of the pocket and are in van der Waals interactions with the base moieties. The Trp 33H side-chain is placed in parallel to the 3'-pyrimidone base, and the Tyr 100iH side-chain is nearly perpendicular to the 5'-thymine base. His 27dL, Tyr 32L, Leu 93L, and Ser 58H forming another side-wall are located in the vicinity of the sugar-phosphate backbone. In the 64M-2 Fab complex with dTT(6-4)TT, 5'- and 3'-side phosphate groups are also involved in interaction with Fab residues.     

Murine polyspecific antibodies. I. Monoclonal and serum anti-DNA antibodies cross-reactive with 2,4,6-trinitrophenyl derivatives

Six anti-DNA hybridoma autoantibodies were prepared by fusing spleen cells from unimmunized MRL/MpJ/lpr/lpr female mice with BALB/c myeloma cells. The monoclonal antibodies were analyzed by solid-phase ELISA for antigen-binding specificities. Three antibodies (62A2, 85A5, and 43B2) bound ssDNA, TNP-KLH, and recognized an epitope(s) present on insolubilized proteins such as BSA, KLH, ferritin, and insulin. The antibodies bound, with a marked preference, TNP-KLH, either soluble or insoluble. The other three antibodies (35A1, 32C5, and 39D2) bound only ssDNA. However, this binding was inhibited by free flavinic acid. None of the six antibodies bound either cardiolipin or proteoglycans, indicating that they do not recognize the repeating negatively charge units common to cardiolipin, proteoglycans, and DNA. All six monoclonal antibodies were purified by affinity chromatography with TNP-Sepharose. Moreover, both anti-DNA and anti-TNP antibodies from sera of nonautoimmune and autoimmune mice were purified easily on TNP-Sepharose.     

Molecular analysis of the autoantibody response in peptide-induced autoimmunity

Immunization of nonautoimmune BALB/c mice with multimeric DWEYSVWLSN, a peptide mimotope of DNA, induces anti-DNA and other lupus-associated Abs. To further investigate the pathogenesis of the autoantibody response induced by peptide immunization, we generated hybridomas from peptide-immunized mice that bound peptide, dsDNA, cardiolipin, Sm/ribonucleoprotein (RNP), or some combination of these Ags. Analysis of 24 IgM Abs led to the identification of three groups of Abs: 1) Abs reactive with peptide alone, 2) anti-peptide Abs cross-reactive with one or more autoantigens, and 3) autoantibodies that do not bind to peptide. The gene families and particular VH-VL combinations used in those hybridomas binding DNA were similar to those used in the anti-DNA response in spontaneous murine lupus. Another similarity to the spontaneous anti-DNA response was the generation of arginines in the complementarity-determining region-3 of DNA-binding hybridomas. Interestingly, one Ab had the VH-VL combination present in the original R4A anti-DNA Ab used to select the DWEYSVWLSN peptide from a phage display library. Many of the heavy and light chains displayed evidence of somatic mutation, suggesting that they were made by Ag-activated B cells. Analysis of the Ab repertoire in peptide-induced autoimmunity may provide insights into the generation of anti-DNA Abs following exposure to foreign Ag. Furthermore, the recovery of an Ab with the heavy and light chain combination of the Ab originally used to isolate the immunizing peptide confirms the utility of phage display peptide libraries in generating true molecular mimics.     

Nature of double-stranded DNA binding activity in seropositive rheumatoid arthritis: formation of low avidity DNA/rheumatoid factor/IgG/low density lipoprotein complexes

Sera from majority of patients with seropositive rheumatoid arthritis, which generally lacked detectable anti-double stranded DNA in Farr, Crithidia luciliae, and microcomplement fixation assays, exhibited high levels of dsDNA binding in the presence of 3.5% polyethylene glycol when using intrinsically labeled 3H-PM2 DNA as antigen. Except for SLE, such increased dsDNA binding was absent in normal and a variety of other disease sera, including those from patients with seronegative rheumatoid arthritis. In contrast to the situation in SLE, in which dsDNA binding is mediated by specific anti-DNA antibody, the increased dsDNA binding activity in seropositive rheumatoid arthritis was shown to be dependent upon complex low avidity interactions involving DNA, IgG, IgM rheumatoid factor, and low density lipoproteins. Analysis of the composition of the polyethylene glycol serum precipitates by 2-dimensional gel diffusion, immunoelectrophoresis, and sodium dodecyl sulfate polyacrylamide gel electrophoresis failed to reveal the presence of additional DNA-binding proteins unique to seropositive rheumatoid arthritis. The only feature distinguishing high DNA binding sera from those with low DNA binding activity was an increased amount of polyethylene glycol-insoluble IgG in the former, presumably reflecting IgG/IgG and/or IgG/IgM complexes. The significance of these unusual DNA/low density lipoprotein/IgG/rheumatoid factor complexes with respect to the diagnostic specificity and pathophysiology of the DNA/anti-DNA system is discussed.     

Identification of chimpanzee Fab fragments by repertoire cloning and production of a full-length humanized immunoglobulin G1 antibody that is highly efficient for neutralization of dengue type 4 virus

A safe and effective dengue vaccine is still not available. Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative for the prevention of dengue virus infection. Fab monoclonal antibodies to dengue type 4 virus (DENV-4) were recovered by repertoire cloning of bone marrow mRNAs from an immune chimpanzee and analyzed for antigen binding specificity, V(H) and V(L) sequences, and neutralizing activity against DENV-4 in vitro. Fabs 5A7, 3C1, 3E4, and 7G4 were isolated from a library constructed from a chimpanzee following intrahepatic transfection with infectious DENV-4 RNA. Fabs 5H2 and 5D9, which had nearly identical V(H) sequences but varied in their V(L) sequences, were recovered from a library constructed from the same chimpanzee after superinfection with a mixture of DENV-1, DENV-2, and DENV-3. In radioimmunoprecipitation, Fab 5A7 precipitated only DENV-4 prM, and Fabs 3E4, 7G4, 5D9, and 5H2 precipitated DENV-4 E but little or no prM. Fab 3E4 and Fab 7G4 competed with each other for binding to DENV-4 in an enzyme-linked immunosorbent assay, as did Fab 3C1 and Fab 5A7. Fab 5H2 recognized an epitope on DENV-4 that was separate from the epitope(s) recognized by other Fabs. Both Fab 5H2 and Fab 5D9 neutralized DENV-4 efficiently with a titer of 0.24 to 0.58 micro g/ml by plaque reduction neutralization test (PRNT), whereas DENV-4-neutralizing activity of other Fabs was low or not detected. Fab 5H2 was converted to full-length immunoglobulin G1 (IgG1) by combining it with human sequences. The humanized chimpanzee antibody IgG1 5H2 produced in CHO cells neutralized DENV-4 strains from different geographical origins at a similar 50% plaque reduction (PRNT(50)) titer of 0.03 to 0.05 micro g/ml. The DENV-4 binding affinities were 0.42 nM for Fab 5H2 and 0.24 nM for full-length IgG1 5H2. Monoclonal antibody IgG1 5H2 may prove valuable for passive immunoprophylaxis against dengue virus in humans.     

Ig H and L chain contributions to autoimmune specificities

An Ig H chain expression vector has been constructed by using the V region of 3H9, an antibody that binds ssDNA, dsDNA, and cardiolipin. The H chain construct was transfected into six hybridoma cell lines expressing Ig L chains. All resulting H and L chain combinations had at least some affinity for ssDNA, whereas five also bound dsDNA to a similar degree as 3H9. The loss of dsDNA binding was correlated with a single amino acid difference between two V kappa 8 L chains. A further characteristic of 3H9, its immunofluorescent staining pattern, was shared by four of the recombinant antibodies, whereas its specificity for cardiolipin was shared with five. The transfections reported here show that a V kappa 3 L chain confers specificity for an RNA-associated epitope and that a V kappa 21E L chain prevents cardiolipin binding. These experiments suggest that the 3H9 H chain contributes essential determinants required for binding to DNA as well as cardiolipin but that L chains can modulate or prevent this binding. L chains may also expand the specificity of a recombinant antibody.     

Human anti-DNA autoantibodies and induced antibodies against ROS-modified-DNA show similar antigenic binding characteristics.

Alterations in DNA structure by hydroxyl radical modification was characterized by UV spectroscopy, Tm, nuclease S1 digestibility and base modification. In view of indicted role of oxygen free radicals in human diseases, an attempt has been made to precisely compare the antigen binding properties of induced antibodies against hydroxyl radical modified DNA with those of naturally occurring anti-DNA autoantibodies. Antibodies induced against ROS-DNA showed diverse antigen binding characteristics which were comparable with those derived from SLE patients. The immune IgG recognized native DNA, heat denatured DNA, and synthetic polynucleotides in B-/B-like conformations. IgG isolated from SLE sera showed preference for ROS-DNA in competition-inhibition assay. The antigenic diversity of induced antibodies and preference of circulating anti-DNA autoantibodies for ROS-DNA over that of native DNA demonstrates the possible role of modified DNA antigens in the pathogenesis of SLE.     

Thermally inactivated simian virus 40 tsA58 mutant T antigen cannot initiate viral DNA replication in vitro.

The mutation in the temperature-sensitive tsA58 mutant T antigen (Ala-438----Val) lies within the presumptive ATP-binding fold. We have constructed a recombinant baculovirus that expresses large quantities of the tsA58 T antigen in infected insect cells. The mutant T antigen mediated simian virus 40 origin-containing DNA (ori-DNA) synthesis in vitro to nearly the same extent as similar quantities of wild-type T antigen at 33 degrees C. However, if wild-type and tsA58 T antigens were heated at 41 degrees C in replication extracts prior to addition of template DNA, the tsA58 T antigen but not the wild type was completely inactivated. The mutant protein displayed greater thermosensitivity for many of the DNA replication activities of T antigen than did the wild-type protein. Some of the replication functions of tsA58 T antigen were differentially affected depending on the presence or absence of ATP during the preheating period. When tsA58 T antigen was preheated in the presence of ATP at 41 degrees C for a time sufficient to completely inactivate its ability to replicate ori-DNA in vitro, it displayed substantial ATPase and normal DNA helicase activities. Conversely, when preheated in the absence of nucleotide, it completely lost both ATPase and helicase activities. Preheating tsA58 T antigen, even in the presence of ATP, led to drastic reductions in its ability to bind to and unwind DNA containing the replication origin. The mutant T antigen also displayed thermosensitivity for binding to and unwinding nonspecific double-stranded DNA in the presence of ATP. Our results suggest that the interactions of T antigen with ATP that are involved in T-antigen DNA binding and DNA helicase activities are different. Moreover, we conclude, consistent with its phenotype in vivo, that the tsA58 T antigen is defective in the initiation but not in the putative elongation functions of T antigen in vitro.