Comparison of poly(A).poly(dT) and poly(I).poly(dC) as immunogens for the induction of antibodies to RNA-DNA hybrids

A goat immunized with poly(A).poly(dT) produced three distinct antibody populations. The major one was specific for RNA-DNA hybrids and was purified from precipitates made with poly(I).poly(dC). It also reacted with hybrids of mixed base composition made with E. coli RNA polymerase. The other populations were purified with poly(A).poly(U) or poly(dT). Three rabbits also produced mainly hybrid-specific antibody in response to poly(A) . poly(dT). A goat immunized with poly(I).poly(dC) formed antibodies reactive with poly(I) and others reactive with poly(dC) but none specific for hybrid structure. Three rabbits did not respond to poly(I).poly(dC). Measurements of reactions with anti-inosine sera, thermal denaturation and sensitivity to S1 nuclease indicated that poly(I).poly(dC) is a less stable helix than poly(A).poly(dT) or poly(I).poly(C). Poly(A).poly(dT) is the more suitable synthetic immunogen for the production of hybrid-specific antibodies.     

Dependence of the antiviral activity of the poly(G).poly(C) complex on the size of the continuous poly(C)segments

Modification of poly(C) by various frequency treatment with adenosine non-complementary to guanosine has produced poly(G) X poly (C.A) complexes with continuous double-stranded areas the length of which is determined by C/A ratio. Studies of the antiviral activity of poly(G).poly(C,A) complexes with C/A from 10:1 to 90:1 and poly(G).poly(C) in vesicular stomatitis virus-infected chick embryo cell cultures and in experimental tick-borne encephalitis of mice demonstrated that the maximum activity is achieved at an average lengths of double-stranded areas of 90 nucleotide pairs. At the same time, a low but statistically significant antiviral activity is observed at a length of double-stranded areas of 10-30 nucleotide pairs.     

A monoclonal antibody to the double-stranded polyribonucleotide complex poly(A) X poly(U)

A monoclonal antibody to the double-stranded polyribonucleotide complex poly(A) . poly(U) was derived from the fusion of spleen cells from immunized DBA/2 mice and the P3 X X63-Ag8 plasma cytoma. Specificity studies using radioimmunoassays showed that the anti-poly(A) . poly(U) does not cross-react with single-stranded polyribonucleotides. RNA X DNA hybrids or DNAs. In addition to RNA duplexes associating adenine and uracil, it recognizes synthetic poly(I) . poly(C) and naturally occurring reovirus RNA. It is thus directed against a conformational epitope with an absolute requirement for two polyribose phosphate chains. However, the antibody does not cross-react with poly(G) . poly(C) and is therefore able to distinguish between RNA double helices.     

Specific binding of poly(I)-poly(C) to the membrane of murine B lymphocyte subsets.

Indirect immunofluorescence revealed that 13% of BALB/c and 33% of CBA spleen cells of B type carry specific binding sites at their surface for double-stranded poly(I).poly(C). Pretreatment of BALB/c spleen cells with anti-mouse immunoglobulin serum increased the number of B cells capable of binding poly(I).poly(C) indicating the existence of a second B lymphocyte subpopulation carrying masked poly(I).poly(C)-binding sites. Pretreatment of the cells with mitogenic doses of either lipopolysaccharide (LPS) or single-stranded polynucleotides, e.g. poly(I) or double-stranded poly(A).poly(U), failed to affect binding of poly(I).poly(C) to the cells. Poly(I).poly(C) converts small poly(I).poly(C)-binding lymphocytes into lymphoblasts carrying poly(I).poly(C)-binding sites. Lymphoblasts derived from LPS-stimulated cells do not carry poly(I).poly(C)-binding sites. Thymocytes or splenic T cells failed to bind poly(I).poly(C). As measured by thymidine uptake, CBA mice containing a higher percentage of poly(I).poly(C)-binding cells, are high responder mice to poly(I).poly(C), compared with low responder BALB/c mice.     

Evaluation of the size of the continuous poly(G) site necessary for the biological activity of the poly(G).poly(C) complex

On the basis of synthesis of a series of poly(G, A).poly(C) copolymers with changing G:A ratio from 15:1 to 90:1 and trials of their biological activity in comparison with poly(G).poly(C), the size of poly(G) in it was evaluated within the range of a continuous double-stranded area necessary for the activity. The antiviral activity close to that of poly(G).poly(C) in experimental tick-borne encephalitis of mice and vesicular stomatitis virus infection of chick embryo cells was found only in poly(G,A).poly(C) complexes with a G:A ratio equal to or higher than 90:1. Consequently, the high activity of poly(G).poly(C) is present at an average length of poly(G) equal to 90-100 nucleotides within the limits of the continuous double-stranded area.     

Are cytotoxicity and interferon inducing activity of poly(I).poly(C) invariably linked in interferon-treated L cells.

Interferon-treated L cells exhibit a specific enhanced susceptibility to the cytotoxic and interferon inducing activities of double-stranded RNAs such as poly(1). poly(C). These activities remained closely linked through widely varying assay conditions, involving, for example, different time anddosage schedules of poly(1). poly (C),suggesting that there is at least one common step in the mechanisms leading to interferon formation and toxicity in interferon-primed cells exposed to poly(1).poly(C). However, some procedures such as addition of metabolic inhibitors (actinomycin D, cycloheximide) and repeated administration of poly(1).poly(C) suppressed the interferon inducing capacity of poly(1).poly(C) without a concomitant decrease of toxicity. Other procedures such as brief treatment of the cells with interferon or DEAE-dextran permitted full expression of the interferon inducing activity of poly(1).poly(C) without any sign of toxicity. The latter results suggest that the mechanisms underlying interferon production and toxicity of poly(1).poly(C) in interferon-treated L cells diverge from a certain point onward.     

Stimulation of B cells by poly A:poly U and poly I:poly C in vitro

Both poly I:poly C and poly A:poly U stimulate [³H]thymidine incorporation by spleen cells derived from normal or from cortisone-treated mice. The highest rate of stimulation was detected in the spleen cells of nude mice. Compared with spleen cells, thymus cells and cortisone-resistant thymus cells gave only a small but reproducible response. The results suggest that double-stranded polynucleotides act preferentially on B cells in the same way as dextran sulphate.     

Evidence for secondary structure in poliovirus virion RNA demonstrated by antibodies against double-stranded RNA.

Poliovirus particle RNA has been considered to have little secondary structure. Specific binding of poliovirion RNA by antibodies against double-stranded RNA (dsRNA) has been confirmed and further characterized by a radioimmunoassay using Staphylococcus aureus protein A to precipitate the nucleic acid-antibody complex. Competitive binding studies between virion single-stranded RNA (ssRNA) and poly(I).poly(C) demonstrated that the dsRNA effectively inhibited binding of radiolabelled poliovirion RNA by the anti-dsRNA antibodies but the virion RNA was a poor competitor of radiolabelled ds RNA. This indicates that both RNAs reacted with the same species of antibodies in the sera, but avidity of the antibodies for dsRNA was greater than for the poliovirion RNA.     

Selective inhibition of hepatitis B virus and human immunodeficiency virus sequence-promoted gene expression by cotransfected poly(I):poly(C)

The transient expression of hepatitis B virus (HBV) surface and "eJ" antigens caused by transfection of human hepatoblastoma HepG2 cells with HBV DNA was markedly inhibited by cotransfection with poly(I):poly(C). Cotransfection with poly(I):poly(C) also inhibited the expression of bacterial chloramphenicol acetyltransferase (CAT) gene which was under the control of either the HBV core promoter or the human immunodeficiency virus (HIV-1) long terminal repeat. This inhibition was much more pronounced on the expression of HBV-promoted CAT than HIV-promoted CAT. The uptake of reporter plasmid was not affected by cotransfected poly(I):poly(C). The inhibition was found to be at the steady-state CAT mRNA level and appeared to be specific for HBV and HIV regulatory sequences since CAT expression directed by other viral and cellular regulatory sequences was not inhibited. Cotransfection with a mixture of equal amounts of poly(I) and poly(C) had similar inhibitory effects whereas cotransfection with poly(l) or poly(C) alone, or other double-stranded ribo- or deoxyribonucleotides, did not have such strong effects. The addition of poly(l):poly(C) to the culture medium of cells transfected with these reporter plasmids caused little inhibition. Transfection with poly(l):poly(C) induced a minimal amount of intracellular interferon-alpha in HepG2 cells which may be involved in selective inhibition of HBV-and HIV-1-directed gene expression. 2-Aminopurine, an inhibitor of double-stranded RNA activated protein kinase known to block interferon gene induction by poly(l):poly(C), partially reversed the poly(l):poly(C)-induced inhibitory effect on HBV-CAT expression.     

Comparative antiviral and interferonogenic activity of synthetic polyribonucleotide complexes of poly(I).poly(C) and poly(G).poly(C) in different cell systems]

The antiviral and interferon-inducing activity of synthetic polyribonucleotide complexes poly(I)-poly(C) and poly(G)-poly(C) was studied in chick embryo, mouse embryo and rabbit kidney cell cultures. In chick embryo cell cultures both polyribonucleotides had similar antiviral activities. The interferon-inducing activity was more marked in poly(G)-poly(C) than in poly(I)-poly(C). In the other two cell cultures poly(I)-poly(C) was considerably superior in both activities. The revealed differences in the comparative activity of the polyribonucleotides in relation to the kind of tissue culture were not associated with differences between them in toxicity, sensitivity to pancreatic RN-ase or with possible differences in the duration of the contact with cells necessary for the achievement of the antiviral effect.     

Activation of the interferon-inducible (2'-5') oligoadenylate synthetase by the Epstein-Barr virus RNA, EBER-1.

The 2'-5' oligoadenylate synthetases and the protein kinase PKR are both interferon-induced, double-stranded RNA-dependent proteins that play important roles in the antiviral effects of the interferons and in cellular growth control. Both enzymes are activated by natural or synthetic dsRNAs and by single-stranded RNAs that possess extensive secondary structure. This report describes the effects of the small Epstein-Barr virus-encoded RNA EBER-1 on the regulation of 2-5(A) synthetase activity. We demonstrate that EBER-1 RNA binds to and activates the human 40-kDa 2-5(A) synthetase in a dose-dependent manner. The efficiency of EBER-1 as an activator of 2-5(A) synthetase is approximately 25% of that of the synthetic double-stranded RNA poly(I)/poly(C), and poly(I)/poly(C) further stimulates enzyme activity even in the presence of a high concentration of EBER-1. Conversely, EBER-1 neither stimulates nor inhibits 2-5(A) synthetase that has been activated by a high concentration of poly(I)/poly(C). Competitive binding assays suggest that the relative affinity of the enzyme for poly(I)/poly(C) is considerably higher than that for EBER-1. Our data indicate that EBER-1, like VAI RNA of adenovirus, TAR RNA of HIV-1, and Rex-RE RNA of HTLV-1, is able to activate the 2-5(A) synthetases. The significance of why several viruses may activate the 2-5(A) synthetase/RNase L-mediated RNA degradation pathway is discussed.     

The reduction of voluntary physical activity after poly I:C injection is independent of the effect of poly I:C-induced interferon-beta in mice

One characteristic of sickness behavior in mice is demonstrated by a reduction in voluntary wheel-running activity during infection. Among synthetic double-stranded (ds) RNAs, polyriboinosinic: polyribocytidylic acid (poly I:C) activates to produce interferon (IFN) -beta, which plays an important role in anti-viral activity and host-defense. However, how voluntary wheel-running activity is regulated during poly I:C infection is unknown. To determine whether poly I:C-induced IFN-beta production is responsible for reduced spontaneous physical activity, we measured poly I:C-induced changes in voluntary wheel-running activity in mice. In this experiment, the mice were injected with poly I:C (0-5 mg/kg i.v.) and/or anti-IFN-beta neutralizing antibody (1.5x10(5) U/kg i.v.). We also observed the direct effect of injection of recombinant IFN-beta (rIFN-beta: 5.0x10(4) and 2.5x10(5) U/kg) on wheel-running behavior. Poly I:C treatment dose-dependently reduced wheel-running activity, and induced an increase in plasma IFN-beta in mice. However the activity was not attenuated by the neutralizing antibody specific to IFN-beta treatment. Additionally, the wheel-running activity in rIFN-beta treated mice was maintained, although they showed a higher IFN-gamma inducible protein (IP)-10 concentration in plasma compared with that of the vehicle group. Our results suggest that the transient reduction in physical activity after poly I:C injection is induced dose dependently, but that the mediator might not be poly I:C-induced IFN-beta.     

Effect of the size of the continuous poly(G) site in poly (G, A).poly (C) complexes on their interferon-inducing activity and their capacity to stimulate the development of immunity

It was established that the level of interferon-inducing activity of poly(G90A1).poly(C) complex in cell cultures and in mice was comparable to that of poly(G).poly(C). As the size of the continuous sites of poly(G) in the purine strand in poly(G, A).poly(C) complexes decreased to 60 and 28 nucleotides, the interferon-inducing activity decreased progressively, was still marked, or approached the zero at G:A ratios equal to 17:1 or 10:1, respectively. All this indicates that the cell receptors responsible for switching on of the induction mechanisms for interferon synthesis still recognize the stimulus 17 and possibly less so stimulus 10 of successively located guanosine nucleotides complementary to poly(C) and provide for the highest interferon production level when their number is equal to or exceeds 90-100. With the exception of poly(G10A1).poly(C) in which the interferon-inducing activity did not exceed its detection threshold, all complexes enhanced noticeably or markedly specific immune response in mice to tick-borne encephalitis after immunization with inactivated unadsorbed tissue culture vaccine against this infection. The level of this immunostimulating activity correlated irregularly with the intensity of their interferon-inducing activity.     

The specificity of antibodies to double-stranded (ds) RNA in antisera prepared to three distinct cytoplasmic polyhedrosis viruses.

Antibodies to double-stranded (ds) RNA were detected in sera of rabbits immunized with virions of three distinct types of cytoplasmic polyhedrosis viruses (CPV). Using a radioimmunoassay method, the antibodies (both IgG and IgM classes) cross-reacted with heterologous ds RNAs which differed in the individual molecular weights of the genome segments, and/or base composition. In competitive binding studies it was shown that the ds RNA antibodies in antisera to all three CPV types could be absorbed by the heterologous RNAs. In addition, the antibodies were completely absorbed by the synthetic double-stranded polyribonucleotide poly(rI:rC). These results showed that the ds RNA antibodies in the antiviral sera were not species specific. In contrast, antibodies prepared to poly(rI:rC) showed some specificity for the homologous antigen. None of the sera cross reacted with single-stranded RNA or DNA.     

A novel trait of naturally occurring anti-DNA antibodies: dissociation from immune complexes in neutral 0.3-0.5 M NaCl

Monoclonal and polyclonal anti-DNA antibodies from autoimmune mice, and experimentally induced rabbit anti-nucleic acid polyclonal antibodies were tested for stability of binding to nucleic acids in the presence of various concentrations of NaCl by an enzyme-linked immunosorbent assay (ELISA). Murine monoclonal antibodies 2C10 (IgG2b) and 1A2 (IgG2a), which are known to react specifically with double-stranded (ds) DNA, dissociated completely from their complexes with DNA when washed with a neutral 0.5 M NaCl solution. Another monoclonal antibody (MoAb) (IgM,kappa), polyreactive with single-stranded (ss) DNA, cardiolipin, and trinitrophenylhapten (TNP), was also dissociated from its complexes with ss DNA, but not from its complexes with TNP, by 0.3-0.5 M NaCl. Similar differences were observed in the binding stability of serum antibodies from autoimmune mice to DNA and TNP. In contrast, anti-nucleic acid polyclonal antibodies induced in rabbits by immunization with poly(I), poly(dT) or poly(ADP-ribose) were not significantly dissociated from their immune complexes with relevant antigens or DNA by 0.5 M NaCl. The finding that nucleic acid antigens were not detached from a solid phase by washing with 0.5 M NaCl solution indicated that the reduction of binding of anti-DNA antibodies in both MoAbs and naturally occurring antibodies was really due to dissociation of the antibodies from immune complexes. This is the first demonstration that DNA epitopes recognized by naturally occurring antibodies in both SLE and its mouse models are sensitive to neutral NaCl concentrations. This novel trait of naturally occurring antibodies will be very useful in studies on the nature of immune complexes in sera and kidneys of cases of systemic lupus erythematosus (SLE).     

The immobilization of glucose oxidase onto radio-frequency plasma-modified poly(etherurethaneurea).

Glucose oxidase was covalently immobilized onto a radio-frequency plasma-modified poly(etherurethaneurea). Thin (90-100 nm) plasma-polymerized N-vinyl-2-pyrrolidone films were deposited onto poly(etherurethaneurea) films. Active sites for the immobilization were obtained via reduction with aqueous sodium borohydride and activation with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate. Modified poly(etherurethaneurea) films were assayed for binding and activity of the immobilized glucose oxidase layer. The results of a modified radioimmunoassay and an 'immunochemical stain' indicated that washing in 900 ml of continuously stirred 2% sodium dodecyl sulfate, 2% Triton X-100, and 20 mM sodium phosphate, pH 7.0, for 24 h each at 4 degrees C was necessary to remove physically adsorbed glucose oxidase from the solid supports. An amperometric activity determination in 9 ml of well-stirred 20 mM sodium phosphate-0.1 M sodium chloride, pH 7.4, gave a qualitative demonstration of the activity of the immobilized enzyme on 18.75 cm2 of modified poly(etherurethaneurea) film. A colorometric activity determination using the coupled reaction with o-dianisidine and peroxidase indicated that glucose oxidase covalently immobilized on approximately 2.4 cm2 of modified poly(etherurethaneurea) film had an activity approximately equal to that of 13.4 nM glucose oxidase in 50 mM sodium acetate, pH 5.1, with a specific activity of approximately 32.0 U/mg at pH 5.1 and room temperature.     

Failure of synthetic polynucleotides to affect the immunogenicity of mycobacterial ribonucleic Acid and ribosomal protein preparations

The synthetic polyribonucleotides adenylic acid (poly A), uridylic acid (poly U), cytidylic acid (poly C), and inosinic acid (poly I), whether single- or double-stranded (poly A:U, poly I:C), cannot replace mycobacterial ribonucleic acid (RNA) in the production of a high immune response in CF-1 mice against tuberculous disease. These conclusions are based on the results of several types of experiments. (i) Poly A and poly U, used either singly or in combination, did not increase the immunogenicity of mycobacterial RNA preparations whether emulsified in Freund's incomplete adjuvant (FIA) or not emulsified. (ii) Mycobacterial ribosomal protein, extracted with 2-chloroethanol, was not immunogenic; the addition of poly A:U to the protein did not produce an immune response and FIA did not affect these results. (iii) The RNA left after the protein was extracted was partially immunogenic when emulsified in FIA even though it was partially degraded. (iv) Mycobacterial RNA prepared with ethyl alcohol and partially degraded with ribonuclease had a significantly lower immunogenic activity, and the original higher immune response was not restored by the addition of poly A:U. (v) Mycobacterial RNA totally degraded by weak alkali was not immunogenic, the original immunogenic activity was not restored by the addition of poly A:U or poly I:C, and FIA again did not influence the results. These findings suggest that (i) protein, polypeptides, or other antigenic fragments, if present, are not the specific immunogens; and (ii) mycobacterial RNA is responsible for the high immunogenic activity of mycobacterial ribosomal and RNA preparations. In addition, since the double-stranded forms of these synthetic polynucleotides markedly potentiate the formation of circulating antibodies, these results also reemphasize the lack of correlation between conventional antibody formation and immunity against tuberculosis.     

Interferon-beta,but not tumor necrosis factor-alpha,production in response to poly I:C is maintained despite exhaustive exercise in mice

It remains unclear whether immune response to viral infection is inhibited by severe exercise. We determined whether exhaustive exercise inhibits interferon (IFN)-β and tumor necrosis factor (TNF)-α production after injection of synthetic double-stranded (ds) RNAs, a polyriboinosinic polyribocytidylic acid (poly I:C), as viral infection model. Male C3H/HeN mice, which were divided into exhaustive-exercised and non-exercised groups, were injected with poly I:C (5 mg/kg). Although TNF-α in response to poly I:C was significantly inhibited by exhaustive exercise, IFN-β was no different in both groups. In in-vitro experiments, catecholamines inhibited poly I:C-induced TNF-α, but not IFN-β, production in macrophages. These results suggest that anti-virus cytokine IFN-β in response to poly I:C might be maintained despite severe stressful exercise.