Suppression of Penaeus merguiensis densovirus following oral delivery of live bacteria expressing dsRNA in the house cricket (Acheta domesticus) model.

Penaeus merguiensis densovirus (PmergDNV) is a serious pathogen of the banana prawn, Penaeus merguiensis leading to at least 28% production loss due to reduced growth rates and mortality of juveniles. In the present study, we reduced PmergDNV titres and subsequent mortality by feeding Acheta domesticus (previously determined as an appropriate animal model for P. merguiensis) with dsRNA specific to the capsid protein by mixing it into their food. Feeding A. domesticus with PmergDNV-specific dsRNA in advance of viral challenge increased their longevity, decreased mortality by 84.4% and reduced viral loads 24-fold below the threshold level required for mortality. Mortalities and viral loads were significantly (both P < 0.001) lower in treatments challenged with PmergDNV following exposure to bacterially expressed PmergDNV-dsRNA. This is the first study to demonstrate gene silencing via RNAi against PmergDNV in vivo through oral administration of live bacteria expressing dsRNA in a model system.

Optimizing cancer treatments to induce an acute immune response: radiation Abscopal effects, PAMPs, and DAMPs.

Clinical results indicate improved survival in poorly differentiated prostate cancer patients following a treatment schedule that maximizes hormone therapy prior to radiation. This may be because of a systemic immune response, called an abscopal effect. A literature review showed an association between acute infection and abscopal cancer remission. This led to the theory that, in the presence of endogenous cancer-specific antigens exposed by cancer necrosis, an innate immune response can adapt to respond to those antigens via a cross-talk mechanism. This theory was validated in an animal model. An acute innate immune T-cell response was stimulated using cluster vaccination with Poly(I:C). In the presence of exogenous cancer-specific antigens, this immune response became adaptive, creating an abscopal effect that resulted in cancer resolution. These concepts may be of clinical value, improving outcomes by inducing systemic abscopal effects.

Targeting bacterial RNA polymerase σ70 for development of broad-spectrum antisense antibacterials.

Bacterial DNA-dependent RNA polymerase (RNAP) is the central enzyme among the progress of transcription. The ubiquity, structural and functional similarities among different bacterial species make it an attractive target for developing protein-level anti-RNAP bactericidal agents with broad spectrum. However, the practical value of RNAP inhibitors is limited by an extensively observed single mutation in rpoB gene (encoding the ß subunit of RNAP), resulting in completely resistant strains. Antisense antibacterials (also called RNA silencers in bacteria) may present an unusual opportunity for developing broad-spectrum sequence-selective nucleic acid based therapeutics. This review will first present a brief state of knowledge on progress and problems in targeting RNAP. Special emphasis will then be given to introduce the advantageous features of RNAP σ70 as a potential target for broad-spectrum antisense inhibition. Characteristics of the antisense antibacterial strategy (including antisense mechanism, basic chemistry involved in nucleic acid analogs, their anti-infection applications in vitro and in vivo) will also be thoroughly described. Notably, our exploration on targeting σ70 gene in gram-negative and gram-positive bacteria respectively for realization of broad-spectrum antisense bactericidal effect will be elaborated on. This firstly demonstrates peptide nucleic acid (PNA)-peptide conjugate as a successful example of broad-spectrum antisense antibacterial. At the end, we will also highlight a few promising targets and delivery strategies that favor the possible development of broad-spectrum antisense antibacterials.

Comparison of different live vaccine strategies in vivo for delivery of protein antigen or antigen-encoding DNA and mRNA by virulence-attenuated Listeria monocytogenes.

Listeria monocytogenes can be used to deliver protein antigens or DNA and mRNA encoding such antigens directly into the cytosol of host cells because of its intracellular lifestyle. In this study, we compare the in vivo efficiencies of activation of antigen-specific CD8 and CD4 T cells when the antigen is secreted by L. monocytogenes or when antigen-encoding plasmid DNA or mRNA is released by self-destructing strains of L. monocytogenes. Infection of mice with self-destructing L. monocytogenes carriers delivering mRNA that encodes a nonsecreted form of ovalbumin (OVA) resulted in a significant OVA-specific CD8 T-cell response. In contrast, infection with L. monocytogenes delivering OVA-encoding DNA failed to generate specific T cells. Secretion of OVA by the carrier bacteria yielded the strongest immune response involving OVA-specific CD8 and CD4 T cells. In addition, we investigated the antigen delivery capacity of a self-destructing, virulence-attenuated L. monocytogenes aroA/B mutant. In contrast to the wild-type strain, this mutant exhibited only marginal liver toxicity when high doses (5 x 10(7) CFU per animal administered intravenously) were used, and it was also able to deliver sufficient amounts of secreted OVA into mice. Therefore, the results presented here could lay the groundwork for a rational combination of L. monocytogenes as an attenuated carrier for the delivery of protein and nucleic acid vaccines in novel vaccination strategies.

Priming Th1 immunity to viral core particles is facilitated by trace amounts of RNA bound to its arginine-rich domain.

Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes. When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity. HBcAg contains 5-20 ng RNA/microg protein while nucleotide binding to HBeAg is not detectable. Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity. HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-gamma release by nonimmune spleen cells. The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20-100 microg/mouse) are codelivered with the Ag. Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag. Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency. Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 microg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 microg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity. Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine. Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity.

Induction of T-cell-mediated immunity against MethA fibrosarcoma by intratumoral injections of a bacillus Calmette-Guérin nucleic acid fraction.

MY-1, which consists of DNA and RNA extracted and purified from bacillus Calmette-Guérin (BCG), has been shown to have strong antitumor activity against various experimental tumors. To examine the role of T cells in the antitumor mechanism of MY-1, the effect of MY-1 injection on the development of tumor-specific immunity against MethA fibrosarcoma was investigated. MY-1 injections inhibited tumor growth less effectively in T-cell-deficient nude mice than in normal BALB/c mice. MethA tumor growth was suppressed after inoculation with L3T4-positive lymphocytes from tumor-bearing mice treated with MY-1. MethA-specific delayed-type hypersensitivity was also detected in tumor-bearing mice treated with MY-1. Immunohistochemical analyses showed that many L3T4-positive and a few Lyt2-positive cells infiltrated the regressing tumors. These results indicate that intratumoral MY-1 injections induce a MethA-specific, L3T4-positive cell-mediated, delayed-type hypersensitivity, which is necessary for the tumor regression.

[Double-stranded RNA from phage F6: its interferon-inducing and antiviral properties]. / Dnunitevaia RNK faga F6: interferonindutsiruiushchie i protivovirusnye svoistva.

Double stranded RNA was isolated from bacteriophage phi 6 parasitizing on phytobacteria. Its interferon-inducing and antiviral activities were shown in vitro and in vivo. In the culture of L-929 cells, interferon resistant to heat and acids was synthesized. The interferon could be practically completely neutralized by specific anti-interferon serum. The phage phi 6 preparation dsRNA in the lyophilized form was studied. The preparation retained its biological activity. It was shown that a preparation containing 30 per cent of dsRNA was less toxic than a preparation containing 100 per cent of dsRNA, the difference in the interferon-inducing activity being insignificant.

Chemical analyses and antitumor activity of hydrosoluble substances from Mycobacterium bovis, strain BCG.

Hydrosoluble substances from BCG were prepared by cold water extraction and by hot phenol-water extraction. Chemical analyses revealed that both of them were derived from cytoplasm. The cold water extract (CWE) was effective in the treatment of C3H/He mice which had received an intraperitoneal inoculation of a syngeneic ascites hepatoma, MH134. The growth of a graft in footpad of mastocytoma P815 in CDF1 mice was retarded by intraperitoneal injections of CWE. A peptidoglycan from cell wall prepared by digestion with lysozyme exerted no antitumor activity in the same experimental condition as for the evaluation of antitumor effect of CWE. These results indicate that the antitumor activity of CWE was not due to the presence of a cell wall component.

Protection of mice against encephalomyocarditis virus infection by preparations of transfer RNA.

Preparations of bacterial transfer RNA (tRNA), give dose-dependent protection of mice against encephalomyocarditis (EMC) virus infection at up to I mg tRNA per mouse with maximum response when the tRNA is administered around 6 h before infection. Protection occurs with intraperitoneally and intravenously administered tRNA against infections by both these routes. In some experiments significant protection occurs by single treatments of tRNA up to 24 h after infection with virus doses of I X LD100. Some tRNA preparations of eukaryotic origin do not give significant protection. Protection is not a feature of all species of bacterial tRNA; partially purified valine, tyrosine and phenylalanine tRNAs from Escherichia coli are not protective. tRNA treatment does not induce circulating interferon nor does it 'hypo-reactivate' the protective effect of poly (I).poly (C) treatment of mice. Humoral and cell mediated immune responses do not seem to be involved in tRNA mediated protection since first, cytosine arabinoside treatment does not affect protection by tRNA; second, serum from mice treated with tRNA and an EMC vaccine does not protect other mice against infection, and third, mice that survive normally lethal infections as a result of tRNA treatment are generally just as susceptible to re-infection as previously untreated, uninfected mice. Silica treatment abolishes protection of mice by tRNA implying that macrophages are necessary. However, tRNA does not seem to act by clearance of virus particles since vaccination of mice by inactivated EMC virus is not affected by tRNA treatment. These results are considered in relation to the presence of a tRNA-like structure in EMC virus RNA and protection of mice by other single stranded polynucleotides.

Factors affecting immunogenic activity of mycobacterial ribosomal and ribonucleic acid preparations.

By following careful procedures, mycobacterial ribosomal fractions and ribonucleic acid (RNA) prepared by ethyl alcohol precipitation were obtained which have immunogenic activities similar to the viable attenuated H37Ra cells of Mycobacterium tuberculosis from which they were obtained. This comparison was based on the amount of ribonucleic acid (RNA) present. These preparations consisted of approximately 63% RNA and 37% protein; no deoxyribonucleic acid or polysaccharide was detected by chemical tests. A high correlation was found between the immunogenic activity of a preparation and the per cent increase in hyperchromicity at 260 nm of a ribonuclease-hydrolyzed portion. Final concentrations of sodium dodecyl sulfate higher than 0.25% when used for the preparation of the ribosomal fractions and RNA resulted in significantly lower immune responses and greater variation between experiments. This was not related to the amount of protein present. The stability of the ribosomal and RNA preparations was tested under a variety of conditions. The need for a good protective adjuvant again was shown since mouse serum readily hydrolyzed the RNA. Equal immunity was obtained after immunization by the intraperitoneal and subcutaneous routes; however, no immune response was obtained when the intravenous route was used. Preliminary results with RNA prepared with phenol showed that it was more easily degraded during preparation. This resulted in a lower immune response than was obtained with the RNA prepared with ethyl alcohol.