Feline leukemia virus envelope gp70 of subgroups B and C defined by monoclonal antibodies with cytotoxic and neutralizing functions

Nine murine monoclonal antibodies (MAb) to the envelope proteins of feline leukemia virus (FeLV) are described. Eight MAb are directed to epitopes of the same molecular species of gp70 and the other MAb is directed to the p15E moiety. Six of the gp70 epitopes are discrete; two are closely associated or overlapping. Four anti-gp70 MAb (2 of IgG2A and 2 of IgG2B subclasses) were directly cytotoxic for FeLV-producer lymphoma cells with cat or with rabbit complement (C). Another MAb (IgG2B), which was not cytotoxic alone, specifically and synergistically increased the cytotoxic effects of both IgG2A MAb. Cytotoxic anti-gp70 MAb also had virus-neutralizing capacity; one MAb recognized a determinant common to all FeLV subgroups (A, B, and C), the others recognized gp70 epitopes not present on subgroup A but common to both B and C subgroups. Competitive inhibition of MAb binding was employed to map spatial distributions of the epitopes, and the results fitted a molecule shaped as an incomplete loop. According to the model, epitopes involved with cytotoxic and virus neutralizing antibody functions were closely associated; the region involved is approximately in the center of the molecule, and it contains epitopes that are variably expressed among individual isolates of FeLV derived from different cat lymphoma cell lines.     

Identification of a linear epitope of interferon-alpha2b recognized by neutralizing monoclonal antibodies.

Four monoclonal antibodies (mAbs) directed against the recombinant human interferon-alpha2b (IFN-alpha2b) were used as probes to study the interaction of the IFN molecule to its receptors. The [125I]IFN-alpha2b binding to immobilized mAbs was completely inhibited by IFN-alpha2b and IFN-alpha2a but neither IFNbeta nor IFNgamma showed any effect. Gel-filtration HPLC of the immune complexes formed by incubating [125I]IFN-alpha2b with paired mAbs revealed the lack of simultaneous binding of two different antibodies to the tracer, suggesting that all mAbs recognize the same IFN antigenic domain. Furthermore, the mAbs were also able to neutralize the IFN-alpha2b anti-viral and anti-proliferative activities as well as [125I]IFN-alpha2b binding to WISH cell-membranes. As [125I]mAbs did not recognize IFN exposed epitopes in the IFN:receptor complexes, mAb induction of a conformational change in the IFN binding domain impairing its binding to receptors was considered unlikely. In order to identify the IFN region recognized by mAbs, IFN-alpha2b was digested with different proteolytic enzymes. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation after blotting to poly(vinylidene difluoride) membranes. Data obtained indicated that the smallest immunoreactive region recognized by mAbs consisted of residues 107-132 or 107-146. As this zone includes the sequence 123-140, which has been involved in the binding to receptors, and our mAbs did not show an allosteric behaviour, it is concluded that they are directed to overlapping epitopes located close to or even included in the IFN binding domain.     

Mutation of the IFNAR-1 receptor binding site of human IFN-alpha2 generates type I IFN competitive antagonists

Type I interferons (IFNs) are multifunctional cytokines that activate cellular responses by binding a common receptor consisting of two subunits, IFNAR-1 and IFNAR-2. Although the binding of IFNs to IFNAR-2 is well characterized, the binding to the lower affinity IFNAR-1 remains less well understood. Previous reports identified a region of human IFN-alpha2 on the B and C helices ("site 1A": N65, L80, Y85, Y89) that plays a key role in binding IFNAR-1 and contributes strongly to differential activation by various type I IFNs. The current studies demonstrate that residues on the D helix are also involved in IFNAR-1 binding. In particular, residue 120 (Arg in IFN-alpha2; Lys in IFN-alpha2/alpha1) appears to be a "hot-spot" residue: substitution by alanine significantly decreased biological activity, and the charge-reversal mutation of residue 120 to Glu caused drastic loss of antiviral and antiproliferative activity for both IFN-alpha2 and IFN-alpha2/alpha1. Mutations in residues of helix D maintained their affinity for IFNAR-2 but had decreased affinity for IFNAR-1. Single-site or multiple-site mutants in the IFNAR-1 binding site that had little or no detectable in vitro biological activity were capable of blocking in vitro antiviral and antiproliferative activity of native IFN-alpha2; i.e., they are type I IFN antagonists. These prototype IFN antagonists can be developed further for possible therapeutic use in systemic lupus erythematosus, and analogous molecules can be designed for use in animal models.     

Comparison of augmentation of human natural killer cell cytotoxicity by interferon-alpha subtypes

The capacity of 3 interferon-alpha (IFN-alpha) subtypes, alpha 1, alpha 2 and alpha 4, to augment human natural killer cytotoxicity after exposure in vitro was shown to be dose-dependent and to differ according to subtype. With 10(2) IU/ml, the lowest IFN concentration used, stimulation of NK activity by IFN-alpha 2 was consistently and significantly greater than by IFN-alpha 4 or IFN-alpha 1. An IFN-alpha analogue in which arginine and lysine residues 121 and 122 were replaced by 2 leucines was generated by site-directed in vitro mutagenesis of the IFN-alpha 4 gene; at equivalent concentrations of antiviral activity, this analogue was 10-fold less effective in NK stimulation. There was a lack of correlation between NK-stimulatory and other activities of the IFN-alpha subtypes and the mutant, suggesting that different biological activities may be mediated by different regions of the IFN-alpha molecule.     

Human leukocyte interferon subtypes have different antiproliferative and antiviral activities on human cells

The antigrowth effects of 5 different cloned human leukocyte IFN subtypes (IFN-alpha A, B, C, D, F) and 2 molecular hybrids between them (IFN-alpha AD(Bg1II) and IFN-alpha DA(Bg1II)) were examined on 6 different human cell lines. The results indicate that the interferons sort into two distinct groups: IFN-alpha B, C and F showed comparable antiproliferative activity which was greater than that of IFN-alpha A, D, AD(Bg1II) and DA(Bg1II). The interferons could also be assigned to one of two groups on the basis of their antiviral activity. IFN-alpha A, D and AD(Bg1II) were observed to be more protective than IFN-alpha B, C and F against HSV-2 and EMCV infections, i.e. the relative antiviral efficacies of the cloned IFN subtypes were the reverse of their antiproliferative activities.     

Antigenic determinants of vesicular stomatitis virus: analysis with antigenic variants

Antigenic variants of vesicular stomatitis virus (VSV) serotypes New Jersey and Indiana (VSV-NJ, VSV-Ind) were selected by using a panel of monoclonal antibodies (MAb) specific for the major surface glycoprotein (G-protein). The reactivity of antigenic variants with the panel of MAb confirmed observations made by competitive binding assays that four distinct antigenic sites (A-D)NJ on the VSV-NJ G-protein and four partially overlapping sites (A, B1, B2, C)Ind on the VSV-Ind G-protein are involved in virus neutralization. Furthermore, subregions within the A epitopes of both serotypes were detected by variant analysis. The frequency of variation at most epitopes was 1 in 10(5) for VSV-NJ and 1 in 10(6) for VSV-Ind. The A3 and C determinants of VSV-Ind, however, defined by MAb that exhibited overlap in binding to other epitopes, appeared to be relatively invariant. Multiple mutations may be necessary to abolish antibody binding at these sites. Overlap of the C group of anti-VSV-Ind MAb with the A epitopes was assigned to the A2 subregion, because variants selected with A2 MAb show reduced binding of C MAb. Heterogeneous antisera from a primary immune response could detect differences in reactivity between variants at the A epitopes and wild-type VSV-NJ or VSV-Ind, suggesting the A epitope is immunodominant. Hyperimmune sera could detect a small difference between ANJ and BNJ variants compared to wild-type VSV-NJ, but could not distinguish between VSV-Ind variants and wild-type VSV-Ind.     

Epstein Barr virus/complement C3d receptor is an interferon alpha receptor.

Interferon alpha contains a sequence motif similar to the complement receptor type two (CR2/CD21) binding site on complement fragment C3d. Antibodies against a peptide with the CR2 binding sequence on C3d react with a peptide carrying the IFN alpha CR2 binding motif (residues 92-99) and with recombinant IFN alpha. The IFN alpha-derived peptide, as well as recombinant IFN alpha, inhibits C3bi/C3d interaction with CR2 on the Burkitt lymphoma Raji. The direct interaction of IFN alpha and CR2 is inhibited by polyclonal anti-IFN alpha, anti-CR2 and anti-C3d peptide antibodies as well as by C3bi/C3d, EBV coat protein gp350/220 and IFN but not by IFN gamma. [125I]IFN alpha binding to Raji cells is inhibited by polyclonal anti-IFN alpha and anti-CR2 antibodies, by peptides with the CR2 binding motif and partially by C3bi/C3d. Monoclonal anti-CR2 antibody HB5, but not OKB-7, blocks IFN alpha binding to Raji cells. CR2 or CR2-like molecules may therefore be the major IFN alpha receptors on B lymphocytes.     

Interferon-induced transfer of viral resistance by human B and T lymphocytes

Enriched human B lymphocytes cocultivated with mouse L cells produced human leukocyte interferon (IFN-alpha) and shortly thereafter transferred antiviral activity to the recipient cells (99% inhibition of expected virus yield). In contrast, cocultivation of enriched T-cell populations with mouse L cells resulted in no IFN production or transfer of antiviral activity. In addition, both T and B lymphocytes pretreated with exogenous IFN or stimulated in vitro by mitogens could transfer antiviral activity to human WISH cells. The transfer of antiviral activity was not blocked by antibodies to IFN. The data indicate that both T and B cells can be recruited by IFN to transfer antiviral activity. Thus, once cells are recruited by IFN they can transfer antiviral activity in the absence of IFN and protect cells locally or distally from the site of infection.     

Viral suppression of the interferon system

Type I interferons (IFN-alpha/beta) were originally discovered by their strong and direct antiviral activity [A. Isaacs, J. Lindenmann, Virus interference. I. The interferon, Proc. R. Soc. Lond. B Biol. Sci. 147 (1957) 258-267]. (see review by J. Lindenmann on p. 719, in this issue). Nevertheless, only very recently it was entirely realized that viruses would not succeed without efficient tools to undermine this potent host defense system. Current investigations are revealing an astonishing variety of viral IFN antagonistic strategies targeting virtually all parts of the IFN system, often in a highly specific manner. Viruses were found to interfere with induction of IFN synthesis, IFN-induced signaling events, the antiviral effector proteins, or simply shut off the host cell macromolecule synthesis machinery to avoid booting of the antiviral host defense. Here, we will describe a few well-characterized examples to illustrate the sophisticated and often multi-layered anti-IFN mechanisms employed by viruses.     

Analysis of antigenic domains on natural and recombinant human IFN-beta by the inhibition of biologic activities with monoclonal antibodies

Human IFN-beta (HuIFN-beta) is a biologically potent protein with both antiviral and antiproliferative activities. To understand better its mode of action, a number of murine mAb were developed against a recombinant (serine-17) HuIFN-beta (rHuIFN-beta ser) and screened by ELISA and neutralization of antiviral activity. The panel of antibodies, composed of both IgA and IgG immunoglobulins, were specific for HuIFN-beta and did not crossreact with HuIFN-alpha or gamma. Furthermore, three functionally distinct epitopes (designated as sites I, II, and III) were identified based on mAb neutralization of antiviral and antiproliferative activities of recombinant and natural HuIFN-beta. Antibodies directed to sites I and II neutralized the antiviral and antiproliferative activities of rHuIFN-beta ser, though the antiviral neutralization potency of the mAb to site II was approximately 10-fold less than mAb to site I. Antibodies directed to site I neutralized both recombinant and natural HuIFN-beta, although the antiviral neutralization potency was approximately 10-fold higher against rHuIFN-beta ser than the native protein. The mAb directed to site II did not demonstrate any significant neutralization of the antiviral or antiproliferative activity of natural HuIFN-beta but neutralized a recombinant HuIFN-beta containing the native sequence. Antibodies recognizing site III did not neutralize the biologic activity of either recombinant or natural HuIFN-beta. Thus, three epitopes on HuIFN-beta have been identified, two of which are associated with both antiviral and antiproliferative activities.     

Natural killer (NK) cell activating factor released from murine thymocytes stimulated with an anti-tumor streptococcal preparation, OK-432

Natural killer (NK) activity of mouse splenocytes was significantly augmented when the splenocytes were incubated for 3 to 4 hr with culture supernatants of mouse thymocytes stimulated by OK-432, an antitumor preparation from the Streptococcus pyogenes SU-strain. Antiviral activity was also detected in the culture supernatants, but IL 2 activity was not. When the culture supernatants of thymocytes stimulated by OK-432 were fractionated on a column of Blue Sepharose CL-6B, NK enhancing activity and antiviral activity were observed in partly overlapping fractions that bound to the column. However, the antiviral activity in the Blue Sepharose-bound fraction was neutralized completely by treatment with anti-IFN (alpha, beta) antiserum, whereas significant NK cell enhancing activity was still observed after treatment with anti-IFN (alpha, beta) antiserum. When the Blue Sepharose-bound fraction was subjected to gel filtration, the NK cell enhancing activity was detected in the 25,000 to 35,000 and 40,000 to 67,000 m.w. regions, but antiviral activity was observed in the over 67,000 m.w. region. These results indicate that a new kind of lymphokine, called natural killer cell activating factor (NKAF), distinct from IFN and IL 2, was found. The NKAF was found to have the following properties: its pI value is between pH 5.5 and 6.5, it binds to concanavalin A- and lentil agglutinin-Sepharose, and it is stable with pH 2-24 hr treatment. In addition, NKAF-producing cells were peanut agglutinin (PNA)-thymocytes when thymocytes were fractionated by the agglutination-sedimentation method with the use of PNA.     

Variations of interferon inactivators and/or inhibitors in human serum and their relationship to interferon therapy

Anti-interferon (IFN) activities of normal human sera and sera from cases suspected of viral infection were determined. Of the normal sera tested (57), 65% presented anti-IFN activities against IFN-alpha and/or -beta preparations; 61% against IFN-beta and 32% against IFN-alpha. Of the sera from suspected virus-infected individuals (44), 82% presented anti-IFN activity to IFN-beta and 57% to IFN-alpha with the majority of sera (91%) exerting anti-IFN activities to IFN-alpha and/or -beta preparation. The observed variations of anti-IFN alpha and beta activities of human sera appear to be reproducible and should be taken into consideration in the clinical application of IFN.     

Specific residues within an amino-terminal domain of 35 residues of interferon alpha are responsible for recognition of the human interferon alpha cell receptor and for triggering biological effects

Bovine interferon alpha C (IFN-alpha C) manifest at least 10(5)-fold lower antiviral activity on human cells than on bovine cells (Velan, B., Cohen, S., Grosfeld, H., Leitner, M., and Shafferman, A. (1985) J. Biol. Chem. 260, 5498-5504). By oligonucleotide site-directed mutagenesis within the coding region for the NH2-terminal 44-residue domain of BoIFN-alpha C, we replaced up to 18 residues by the corresponding HuIFN-alpha J1 residues. (HuIFN-alpha J1 is less than 60% homologous in sequence to BoIFN-alpha C.) The nine different bovine-human-IFN alpha hybrids obtained were compared to BoIFN-alpha C and HuIFN-alpha J1 with respect to their potential to induce an antiviral state, synthesis of 2-5A-synthetase, and their specific binding to human and bovine cells. Relative to BoIFN-alpha C, a gradual increase in biological activities (antiviral or 2-5A-synthetase) of approximately 10-, 10(2)-, 10(3)-, and approximately 10(4)-fold is obtained, depending on the number and positions of the residues substituted. A direct correlation exists between biological response and ability of IFN alpha to bind specifically to human cells. A BoIFN alpha molecule mutated in the 10-44 NH2-terminal domain was obtained which is 15, 8, and 35% as active as HuIFN-alpha J1 on human cells in specific binding, induction of antiviral, and 2-5A-synthetase activities, respectively. We concluded that at least 5 of the 12 residues at positions 10; 21, 22, 24; 27; 31, 34, 35, 37, 40; 42, 43 in the 10-44 NH2-terminal domain are critical for recognition of the human IFN-alpha cell receptor and for biological activity. These residues are found among 10 strictly conserved residues in all reported mammalian IFN alpha S, and they act in a cooperative manner to induce a biological response in human cells. The gap between the extent of improvement in binding capacity of the BoIFN alpha mutants on human cells and the corresponding biological response suggests that the primary signal of binding to the cell receptor is amplified within the cell. On bovine cells, HuIFN-alpha J1 and BoIFN-alpha C also compete for the same receptor, and it seems that at least part of the 10-44 NH2-terminal domain on IFN alpha is also involved in interaction with the bovine IFN alpha cell receptor.     

Immunochemical mapping of alpha-2 interferon

A panel of five monoclonal antibodies, designated U1-U5, produced by murine hybridoma clones has been raised to recombinant interferon (IFN) alpha-2, and one monoclonal antibody, designated U6, has been raised to a mixture of cyanogen bromide fragments of IFN alpha-2. These antibodies have been characterized with respect to (1) neutralization of IFN antiviral and antiproliferative activities, (2) binding to four cloned IFN alpha subtypes (alpha-1, alpha-2, alpha-4, and alpha-7) that are naturally occurring and to two novel products of recombinant DNA technology (delta-4 alpha-1 and delta-4 alpha-2/alpha-1 hybrid), and (3) binding to three cyanogen bromide fragments of IFN alpha-2. Four of the six monoclonal antibodies inhibited IFN antiviral activity. In conjunction with the previously reported monoclonal antibodies III/21 [Arnheiter, H., Thomas, R. M., Leist, T., Fountoulakis, M., & Gutte, B. (1981) Nature (London) 294, 278-280] and NK-2 [Secher, D. S., & Burke, D. C. (1980) Nature (London) 285, 446-450], eight unique epitopes have been described. Analysis of cross-reactivity patterns with IFN alpha fragments and subtypes indicated that monoclonal antibodies U1 and NK-2, which neutralized both antiviral and antiproliferative activities, and U2, which was nonneutralizing in these assays, were directed to distinct epitopes located in a polypeptide consisting of the amino-terminal 15 amino acid residues linked to residues 60-110 by a disulfide bond. The epitope recognized by U1 was determined to reside, at least in part, between residues 5 and 15. Competitive binding studies indicated that neutralizing monoclonal antibody U3, which did not bind to any of the cyanogen bromide fragments, was directed to an epitope partially overlapping that of NK-2. Epitopes to which neutralizing monoclonal antibodies U3, U4, and U5 and nonneutralizing antibody U6 were directed were readily distinguished by cross-reactivity with IFN alpha subtypes. The nonneutralizing monoclonal antibody U6 was determined to be directed to an epitope between residues 22 and 58. The fact that delta-4 alpha-1 and the delta-4 alpha-2/alpha-1 hybrid were active in an antiviral assay indicated a lack of direct functional significance for the first four amino-terminal amino acid residues and the Cys1-Cys98 disulfide bond. However, reduction with 2-mercaptoethanol of IFN alpha-2 altered the integrity of four of the eight epitopes. These data support a critical role for disulfide linkages in maintaining the native conformation of IFN alpha-2 and provide a potential basis for predicting the location of functionally important domains.     

Interferon-induced inhibition of receptor-mediated endocytosis of colony-stimulating factor (CSF-1) by murine peritoneal exudate macrophages

Apart from its characteristic antiviral activity, interferon (IFN) also exerts a variety of biologic effects on macrophages. We have studied the effect of IFN on the expression of the colony-stimulating factor receptors (CSF-1 receptors) by murine peritoneal exudate macrophages (PEM). At 37 degrees C, murine IFN decreased the expression of the CSF-1 receptor activity in a time- and dose-dependent fashion by PEM from both endotoxin-sensitive (C3H/Sn) and endotoxin-resistant strains (C3H/HeJ) of mice. Scatchard analysis from the binding data suggests that the decreased expression of CSF-1 receptors is a result of decreased number of receptors rather than a decreased binding affinity. When IFN was incubated with anti-IFN before the addition to cultures, the effect was completely abolished indicating that this activity resides in the same molecules as IFN. The suppressed CSF-1 receptor activity on PEM by IFN appeared to be stable. Removal of added IFN never resulted in a full recovery of CSF-1 binding activity by PEM even after prolonged incubation (7 days). IFN also inhibited the receptor-mediated uptake and utilization of CSF-1 molecules by treated cells, which appeared to be a direct effect of the decreased number of CSF-1 receptors. Treatment of PEM with dexamethasone, prostaglandin, transferrin, insulin, or dibutyryl cAMP failed to suppress both the expression of CSF-1 receptors and CSF-1 utilization by PEM. These studies suggest that IFN may play a role in the regulation of both macrophage production and differentiation via the modulation of specific membrane receptors and inhibition of receptor-mediated CSF-1 endocytosis.     

Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin

We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.     

Probing functional sites on complement protein B with monoclonal antibodies. Evidence for C3b-binding sites on Ba

We used four mouse monoclonal antibodies (Mab) as probes of functional sites of human complement protein B. Two Mab, HA4-1B (gamma 2a kappa) and HA4-15 (gamma 2a kappa), reacted with the same or adjacent epitopes on the Bb fragment of B, while the other two, HA4-1A (gamma 1 kappa) and FD3-20 (gamma 1 kappa), reacted with distinct epitopes on Ba. All reactive epitopes were expressed on native B and only one, recognized by the anti-Ba Mab HA4-1A was more reactive on isolated Ba than on B. These binding specificities were determined by direct binding radioassays and confirmed by inhibition studies. Immunoelectron microscopy of B and Bb in complex with anti-Ba and anti-Bb revealed that the recognized epitopes are on opposite sides of the molecule and are on discrete domains. All four Mab inhibited the hemolytic activity of B, although with different efficiencies and through different mechanisms. The main effect of the two anti-Bb Mab was an increased rate of loss of hemolytic sites from preformed EC3bBb C3 convertase presumably through accelerated dissociation of Bb. On the other hand, the main effect of the two anti-Ba Mab was inhibition of binding of B to C3b. HA4-1A was more efficient, inhibiting by 50% the binding of [125I]B to EC3b at 10 micrograms/ml as IgG and at 13 micrograms/ml as Fab. The data suggest that a binding site for C3b on intact B is located on the Ba portion of the molecule.     

Therapy-induced antibodies against the antiviral and antiproliferative effects of interferons in patients with chronic hepatitis C virus infection

Sera from 86 patients with chronic hepatitis C virus (HCV) infection treated with recombinant interferons-alpha (rIFN-alpha) were screened for IFN-binding and antiviral effect-neutralizing antibodies. Out of the 61 patients treated with rIFN-alpha2b, 46% had binding and 28% had neutralizing antibodies. 44% of the 25 patients treated with rIFN-alpha2a developed binding antibodies and 24% had neutralizing antibodies. Contradictory data were observed concerning the appearance of anti-IFN antibodies and the outcome of IFN therapy. A significantly higher number of the patients with a sustained response to rIFN-alpha2b therapy formed antibodies than the number among the non-responder patients. At the same time, in the patients treated with rIFN-alpha2a, opposite data were found. The activity of the antibodies in some sera was studied against the antiproliferative effect of IFNs on Daudi cells by measuring the [3H]thymidine incorporation. The binding antibodies without neutralization of the antiviral effect of the IFNs inhibited the antiproliferative activity of the rIFNs, similarly to antibodies having both IFN-binding and antiviral effect-neutralizing capacities. At the same time, the antiproliferative effect of the natural IFN was less affected. It is suggested that the antiproliferative assay is more sensitive than the antiviral method for demonstration of the presence of antibodies exerting an inhibitory effect on the biological activities of IFN.     

Divergence of binding, signaling, and biological responses to recombinant human hybrid IFN.

Three human IFN-alpha hybrids, HY-1 [IFN-alpha21a(1-75)/alpha2c(76-165)], HY-2 [IFN-alpha21a(1-95)/alpha2c(96-165)], and HY-3 [IFN-alpha2c(1-95)/alpha21a(96-166)], were constructed, cloned, and expressed. The hybrids had comparable specific antiviral activities on Madin-Darby bovine kidney (MDBK) cells but exhibited very different antiproliferative and binding properties on human Daudi and WISH cells and primary human lymphocytes. Our data suggest that a portion of the N-terminal region of the molecule is important for interaction with components involved in binding of IFN-alpha2b while the C-terminal portion of IFN is critical for antiproliferative activity. A domain affecting the antiproliferative activity was found within the C-terminal region from amino acid residues 75-166. The signal transduction properties of HY-2 and HY-3 were evaluated by EMSA and RNase protection assays. Both HY-2 and HY-3 induced activation of STAT1 and 2. However, HY-2 exhibited essentially no antiproliferative effects at concentrations that activated STAT1 and 2. Additionally, at concentrations where no antiproliferative activity was seen, HY-2 induced a variety of IFN-responsive genes to the same degree as HY-3. RNase protection assays also indicate that, at concentrations where no antiproliferative activity was seen for HY-2, this construct retained the ability to induce a variety of IFN-inducible genes. These data suggest that the antiproliferative response may not be solely directed by the activation of the STAT1 and STAT2 pathway in the cells tested.     

Monoclonal antibodies recognizing amino-terminal and carboxy-terminal regions of human aldolase A: probes to detect conformational changes of the enzyme.

Three monoclonal antibodies (MAbs1A2, 3C5, and 4C2) for human aldolase A [EC 4.1.2.13] were established. MAbs1A2, 3C5, and 4C2 were shown to belong to subclasses IgM, IgG1, and IgG2a, respectively. None of the MAbs inhibits aldolase A activity. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs to chimeric proteins between aldolases A and B [Kitajima et al. (1990) J. Biol. Chem. 265, 17493-17498] in ELISA and to the CNBr-cleaved fragments of aldolase A in immuno-blotting. MAbs1A2 and 3C5 reacted with sites located within amino acid residues 306-363 at the C-terminal region of the enzyme. MAb4C2 recognized an epitope of the enzyme present within amino acid residues 34-108 at the N-terminal region. In a competitive binding assay, MAbs1A2 and 3C5 competed with each other for binding to the antigen and also interfered with the binding of MAb4C2, whereas MAb4C2 failed to inhibit the binding of MAbs1A2 and 3C5 to the antigen. MAb3C5 showed a species-specificity in the reaction with the antigen; it reacted with human and rabbit aldolase A with similar reactivity but not at all with the rat and mouse enzymes, which differ from the human and rabbit enzymes in two amino acid residues at positions 328 and 348. Reactivities of MAbs to aldolase A were further examined with engineered enzymes containing an amino acid substitution.(ABSTRACT TRUNCATED AT 250 WORDS)