Meet the authors: Katalin Karikó and Drew Weissman.

We talk to first and last authors Katalin Karikó and Drew Weissman about their seminal 2005 paper ''Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA", about how they see the work in retrospect, the current progress in the field, and their inspiration-then and now.

Impact of hepatic steatosis on treatment response in nuclesos(t)ide analogue-treated HBeAg-positive chronic hepatitis B: a retrospective study.

BACKGROUND: The impact of hepatic steatosis (HS) on treatment response following nucleos(t)ide analogue (NA) treatment for chronic hepatitis B (CHB) patients has not been clearly elucidated. We aimed to investigate the difference in HBeAg seroclearance between NA-treated HBeAg-positive CHB patients with and without HS. METHODS: We retrospectively recruited HBeAg-positive CHB patients receiving liver biopsy and NA monotherapy. The baseline clinical characteristics and cumulative incidence of HBeAg seroclearance were compared between patients with and without HS and age/gender-matched subgroup analysis was performed. RESULTS: A total of 196 patients were enrolled from 2003 April to 2016 October. The mean age was 39.6 ± 11.2 years, 142 (72.4%) were males and 94 (48%) had histological evidence of HS. Median treatment duration and follow-up period were 24.3 months and 54.9 months, respectively. HBeAg seroclearance was achieved in 56/102 (54.9%) and 54/94 (57.4%) patients with and without HS, respectively (p = 0.830). The 5-year cumulative incidence of HBeAg seroclearance in patients with and without HS was 62.8 and 67.7% in overall population (p = 0.398) and 62.4 and 66.9% in age/gender-matched subgroups (p = 0.395), respectively. The rate of HBeAg seroclearance was comparable between patients with or without HS in different NA monotherapy (all p > 0.05). CONCLUSIONS: HS had no significant impact on HBeAg seroclearance in HBeAg-positive CHB patients with NA monotherapy during long-term follow-up.

Nucleoside-modified mRNA encoding HSV-2 glycoproteins C, D, and E prevents clinical and subclinical genital herpes.

The goals of a genital herpes vaccine are to prevent painful genital lesions and reduce or eliminate subclinical infection that risks transmission to partners and newborns. We evaluated a trivalent glycoprotein vaccine containing herpes simplex virus type 2 (HSV-2) entry molecule glycoprotein D (gD2) and two immune evasion molecules: glycoprotein C (gC2), which binds complement C3b, and glycoprotein E (gE2), which blocks immunoglobulin G (IgG) Fc activities. The trivalent vaccine was administered as baculovirus proteins with CpG and alum, or the identical amino acids were expressed using nucleoside-modified mRNA in lipid nanoparticles (LNPs). Both formulations completely prevented genital lesions in mice and guinea pigs. Differences emerged when evaluating subclinical infection. The trivalent protein vaccine prevented dorsal root ganglia infection, and day 2 and 4 vaginal cultures were negative in 23 of 30 (73%) mice compared with 63 of 64 (98%) in the mRNA group (P = 0.0012). In guinea pigs, 5 of 10 (50%) animals in the trivalent subunit protein group had vaginal shedding of HSV-2 DNA on 19 of 210 (9%) days compared with 2 of 10 (20%) animals in the mRNA group that shed HSV-2 DNA on 5 of 210 (2%) days (P = 0.0052). The trivalent mRNA vaccine was superior to trivalent proteins in stimulating ELISA IgG antibodies, neutralizing antibodies, antibodies that bind to crucial gD2 epitopes involved in entry and cell-to-cell spread, CD4+ T cell responses, and T follicular helper and germinal center B cell responses. The trivalent nucleoside-modified mRNA-LNP vaccine is a promising candidate for human trials.

Nucleoside Modified mRNA Vaccines for Infectious Diseases.

In recent years, numerous studies have demonstrated the outstanding abilities of mRNA to elicit potent immune responses against pathogens, making it a viable new platform for vaccine development (reviewed in Weissman, Expert Rev Vaccines 14:265-281, 2015; Sahin et al., Nat Rev Drug Discov 13:759-780, 2014). The incorporation of modified nucleosides in mRNA has many advantages and is currently undergoing a renaissance in the field of therapeutic protein delivery. Its use in a vaccine against infectious diseases has only begun to be described, but offers advantages for the generation of potent and long-lived antibody responses. FPLC purification and substitution of modified nucleosides in the mRNA make it non-inflammatory and highly translatable (Kariko et al., Immunity 23:165-175, 2005; Kariko et al., Mol Ther 16:1833-1840, 2008; Kariko et al., Nucleic Acids Research 39:e142, 2011) that are crucial features for therapeutic relevance. Formulation of the mRNA in lipid nanoparticles (LNPs) protects it from degradation enabling high levels of protein production for extended periods of time (Pardi et al., J Control Release, 2015). Here, we describe a simple vaccination method using LNP-encapsulated 1-methylpseudouridine-containing FPLC purified mRNA in mice. Furthermore, we describe the evaluation of antigen-specific T and B cell responses elicited by this vaccine format.

Transcriptome Signatures Reveal Rapid Induction of Immune-Responsive Genes in Human Memory CD8(+) T Cells.

Memory T cells (TM) play a prominent role in protection and auto-immunity due to their ability to mount a more effective response than naïve T cells (TN). However, the molecular mechanisms underlying enhanced functionality of TM are not well defined, particularly in human TM. We examined the global gene expression profiles of human CD8(+) TN and TM before and after stimulation. There were 1,284, 1,373 and 1,629 differentially expressed genes between TN and TM at 0 hr, 4 hr and 24 hr after stimulation, respectively, with more genes expressed to higher levels in TM. Genes rapidly up-regulated in TN cells were largely involved in nitrogen, nucleoside and amino acid metabolisms. In contrast, those in CD8(+) TM were significantly enriched for immune-response-associated processes, including cytokine production, lymphocyte activation and chemotaxis. Multiple cytokines were rapidly up-regulated in TM cells, including effector cytokines known to be produced by CD8(+) T cells and important for their functions, as well as regulatory cytokines, both pro- and anti-inflammatory, that are not typically produced by CD8(+) T cells. These results provide new insights into molecular mechanisms that contribute to the enhanced functionality of human CD8(+) TM and their prominent role in protection and auto-immunity.

A deficiency in nucleoside salvage impairs murine lymphocyte development, homeostasis, and survival.

The homeostasis of the immune system is tightly controlled by both cell-extrinsic and -intrinsic mechanisms. These regulators, not all known to date, drive cells in and out of quiescence when and where required to allow the immune system to function. In this article, we describe a deficiency in deoxycytidine kinase (DCK), one of the major enzymes of the nucleoside salvage pathway, which affects peripheral T cell homeostatic proliferation and survival. As a result of an N-ethyl-N-nitrosourea-induced mutation in the last α helix of DCK, a functionally null protein has been generated in the mouse and affects the composition of the hematopoietic system. Both B and T lymphocyte development is impaired, leading to a state of chronic lymphopenia and to a significant increase in the number of myeloid cells and erythrocytes. In the periphery, we found that mutant lymphocytes adopt a CD44(high)CD62L(low) memory phenotype, with high levels of proliferation and apoptosis. These phenotypes are notably the result of a cell-extrinsic-driven lymphopenia-induced proliferation as wild-type cells transferred into DCK-deficient recipients adopt the same profile. In addition, DCK also regulates lymphocyte quiescence in a cell-intrinsic manner. These data establish dCK as a new regulator of hematopoietic integrity and lymphocyte quiescence and survival.

Potential inhibition of somatic hypermutation by nucleoside analogues.

Somatic hypermutation, which occurs in antigen-activated germinal centre B lymphocytes, diversifies the genes that encode immunoglobulin variable regions and leads to the 'affinity maturation' of the humoral immune response. Hypermutation affects dC/dG and dA/dT pairs with approximately equal frequency in vivo. DNA polymerase-theta contributes to hypermutagenesis at dC/dG pairs and DNA polymerase-eta is substantially involved in the generation of hypermutations at dA/dT pairs. The biochemical properties of polymerases-theta and -eta indicate that their DNA synthetic activities are potentially susceptible to inhibition by nucleoside analogues, so it is feasible that nucleoside analogues reduce the accumulation of dC/dG- and dA/dT-targeted hypermutations in vivo. Nucleoside analogues could hence impair the humoral adaptive immune response of HIV-infected patients who are prescribed these chemotherapeutic agents.

Cross-study analysis of the relative efficacies of oral antiviral therapies for chronic hepatitis B infection in nucleoside-naive patients.

BACKGROUND AND OBJECTIVE: Lamivudine and adefovir were approved for treatment of chronic hepatitis B virus (HBV) infection based on placebo-controlled trials, and entecavir was recently approved on the basis of its superiority over lamivudine in phase II/III trials; however, to date, these three therapies have not been compared head to head. METHODS: To evaluate the relative efficacy of these therapies, we applied a predefined protocol of established statistical techniques to compare data from phase III entecavir trials with published clinical trial results with lamivudine, adefovir and placebo in nucleoside-naive hepatitis B e antigen (HBeAg)-positive and -negative populations. RESULTS: A comprehensive literature search identified 612 publications/data sources, of which 28 satisfied predefined inclusion criteria. Independent reviewers extracted week 48-52 histological, virological, biochemical and serological endpoints from these sources, which were analysed with a fixed-effects model. For each of the three histological endpoints in HBeAg-positive patients (Histological Improvement, Ranked Assessment of Necroinflammation [RA-N] and Ranked Assessment of Fibrosis [RA-F]), entecavir was superior to adefovir. Entecavir was superior to lamivudine for Histological Improvement and comparable to lamivudine for RA-N and RA-F. With respect to reducing HBV DNA levels, entecavir (-6.98 log(10) copies/mL) was more effective than lamivudine (-5.46 log(10) copies/mL, p < 0.0001) and adefovir (-3.60 log(10) copies/mL, p < 0.0001), and lamivudine was more effective than adefovir (p < 0.0001). The parallel goals of HBV DNA reduction below the limit of quantitation (LOQ) [by polymerase chain reaction] and ALT normalisation were achieved more often with entecavir (69% and 67% of patients, respectively) than with lamivudine (38% and 59%, respectively; p < 0.0001 and p < 0.05, respectively) or adefovir (21% and 48%, respectively; both p < 0.0001), and more often with lamivudine than with adefovir (p < 0.0001 and p < 0.05, respectively). HBeAg seroconversion rates were higher with entecavir (21% of patients) and lamivudine (18%) than with adefovir (12%, p < 0.01 and p < 0.05, respectively). For each of the three histological endpoints in the HBeAg-negative population, entecavir was comparable to adefovir. Entecavir was superior to lamivudine for Histological Improvement, and comparable to lamivudine for RA-N and RA-F, and all three antivirals were superior to placebo. Entecavir proved superior to lamivudine and adefovir in lowering HBV DNA levels (-5.20 vs -4.66 vs -3.91 log(10) copies/mL, respectively; p < 0.0005 and p < 0.0001, respectively) and in suppressing HBV DNA below the LOQ (91% vs 73% vs 51% of patients, respectively; both p < 0.0001); in the latter respect, lamivudine was in turn superior to adefovir (p < 0.0001). Entecavir was also superior to lamivudine in normalising ALT (76% vs 69% patients, respectively; p < 0.05). CONCLUSIONS: Over a 12-month treatment period, this analysis predicts that the antiviral efficacy of entecavir would be superior to that of lamivudine, which in turn would be superior to that of adefovir, in nucleoside-naive patients with chronic HBV infection.

Generation of double gene disruptions in Dictyostelium discoideum using a single antibiotic marker selection.

Gene targeting is a powerful molecular genetic technique that has been widely used to understand specific gene function in vivo. This technique allows the ablation of an endogenous gene by recombination between an introduced DNA fragment and the homologous target gene. However, when multiple gene disruptions are needed, the availability of only a limited number of marker genes becomes a complication. Here we describe a new approach to perform double gene disruptions in Dictyostelium discoideum by simultaneous transfection of two gene targeting cassettes followed by performing clonal selection against only one marker gene. The subsequent PCR-based screens of blasticidin-resistant clones revealed the integration of both the selected and the nonselected targeting cassettes at their original respective loci creating complete gene disruptions. For the genes we have tested in these studies (myosin heavy chain kinases B and C), the efficiency of the double gene targeting event is found in the range of 2%-5% of all blasticidin-resistant colonies following the transfection step. This approach for the simultaneous disruptions of multiple genes should prove to be a valuable tool for other laboratories interested in creating multiple gene disruptants in Dictyostelium or other organisms where a limited number of selectable markers are available.

Slipped (CTG).(CAG) repeats of the myotonic dystrophy locus: surface probing with anti-DNA antibodies.

At least 15 human diseases have been associated with the length-dependent expansion of gene-specific (CTG).(CAG) repeats, including myotonic dystrophy (DM1) and spinocerebellar ataxia type 1 (SCA1). Repeat expansion is likely to involve unusual DNA structures. We have structurally characterized such DNA, with (CTG)(n).(CAG)(n) repeats of varying length (n=17-79), by high-resolution gel electrophoresis, and have probed their surfaces with anti-DNA antibodies of known specificities. We prepared homoduplex S-DNAs, which are (CTG)x.(CAG)y where x=y, and heteroduplex SI-DNAs, which are hybrids where x>y or x

Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7.

Certain C8-substituted and N7, C8-disubstituted guanine ribonucleosides comprise a class of small molecules with immunostimulatory activity. In a variety of animal models, these agents stimulate both humoral and cellular immune responses. The antiviral actions of these guanosine analogs have been attributed to their ability to induce type I IFNs. However, the molecular mechanisms by which the guanosine analogs potentiate immune responses are not known. Here, we report that several guanosine analogs activate Toll-like receptor 7 (TLR7). 7-Thia-8-oxoguanosine, 7-deazaguanosine, and related guanosine analogs activated mouse immune cells in a manner analogous to known TLR ligands, inducing cytokine production in mouse splenocytes (IL-6 and IL-12, type I and II IFNs), bone marrow-derived macrophages (IL-6 and IL-12), and in human peripheral blood leukocytes (type I IFNs, tumor necrosis factor alpha and IL-12). The guanosine congeners also up-regulated costimulatory molecules and MHC I/II in dendritic cells. Genetic complementation studies in human embryonic kidney 293 cells confirmed that the guanosine analogs activate cells exclusively via TLR7. The stimulation of TLR7 by the guanosine analogs in human cells appears to require endosomal maturation because inhibition of this process with chloroquine significantly reduced the downstream activation of NF-kappaB. However, TLR8 activation by R-848 and TLR2 activation by [S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-R-Cys-S-Ser-Lys4-OH, trihydrochloride)] were not inhibited by chloroquine, whereas TLR9 activation by CpG oligodeoxynucleotides was abolished. In summary, we present evidence that guanosine analogs activate immune cells via TLR7 by a pathway that requires endosomal maturation. Thus, the B cell-stimulating and antiviral activities of the guanosine analogs may be explained by their TLR7-activating capacity.

Monitoring of urinary excretion of modified nucleosides in cancer patients using a set of six monoclonal antibodies.

Monoclonal antibodies were produced and characterized in order to allow the monitoring of the urinary excretion of six modified nucleosides. The specificity of each antibody was determined and competitive solid-phase enzyme-linked immunoassays were designed, the sensitivity of which lay in the pmol range. Detection and quantitation of 5-methylcytidine (5-MeCyd), 4-acetylcytidine (4-AcCyd), 1-methylinosine (1-MeIno), 1-methyladenosine (1-MeAdo), 7-methylguanosine (7-MeGuo) and pseudouridine (psi-Urd) can be performed in small volumes (70 microliters) of untreated urine. Results can be obtained from as many as 20 different samples, for one molecule, within 3 h. With this technique, values observed for three commonly measured nucleosides in urine from healthy subjects (psi-Urd, 1-MeAdo and 1-MeIno) are in good agreement with those reported by other authors after analysis by high performance liquid chromatography. Results obtained in urine from cancer patients show significantly increased levels of the six haptens quantitated by this immunoassay.

Auto antibodies to nucleosides in systemic lupus erythematosus.

Serological studies in 110 patients with systemic lupus erythematosus (SLE) have shown that autoantibodies to DNA and RNA had subspecificity to adenosine (30.9%), cytidine (79%), guanosine (44.5%), thymidine (20%) and uracil (56.3%). It was also observed that DNA antibodies are heterogenous and that antibody with specificity for both the native confirmation as well as exposed nucleoside of the denatured molecule were present in sera of most of the patients with SLE. There was also alteration in the pattern of antibody to nucleoside in some patients who were treated with steroids or immunosuppressive drugs.

Monoclonal anti-nucleoside antibodies. Characterization and application in an enzyme immunoassay of single-stranded DNA.

Two mouse monoclonal antibodies were raised against adenosine and guanosine coupled to bovine serum albumin (BSA) by periodate oxidation. They were named A-16 and G-K21 respectively and selected for their ability to recognize single-stranded DNA. Their epitope specificities were assessed and their dissociation constants determined by an indirect ELISA method. The KD values for adenosine and guanosine coupled to BSA were 9.9 X 10(-7) M and 1.1 X 10(-10) M for G-K21 respectively, and 2.5 X 10(-8) M and 1.0 X 10(-6) M for A-16. These monoclonal anti-nucleoside antibodies were used to develop a sandwich enzyme immunoassay for single-stranded DNA. The purified IgG antibodies were coupled to beta-galactosidase and alkaline phosphatase by the one-step glutaraldehyde method and used in a test optimized for pH, saturating proteins, coating antibody, nature of the conjugate and protein concentrations. Less than 100 pg/well of single-stranded DNA could be detected, and the detection was linear over a DNA concentration ranging from 0.34 to 34 ng/ml. The assay could quantitate single-stranded DNAs of differing origin, but not RNAs. The test was compared to another titration method, and used to calibrate target DNA amounts in non-radioactive hybridization experiments.

Antiguanosine antibodies: a new marker for procainamide-induced systemic lupus erythematosus.

Antinuclear antibodies are present in most patients receiving procainamide. To ascertain whether IgG antiguanosine antibodies are associated with the development of the symptoms of systemic lupus erythematosus, we compared the levels of these antibodies in the sera of 65 patients receiving procainamide: 18 with procainamide-induced symptoms and 47 asymptomatic patients. Antinuclear antibodies measured by immunofluorescence were present in the 18 patients with drug-induced symptoms but also in 24 asymptomatic patients. Similarly, elevated serum levels of antibodies to single-stranded DNA were found in 15 patients with symptoms and in 20 asymptomatic patients. In contrast, levels of IgG antiguanosine antibodies were elevated in 15 patients with drug-induced symptoms, but in only 3 asymptomatic patients. Antiguanosine antibodies binding to single-stranded DNA were found primarily in patients with arthritis, pleuritis, and pericarditis. These results suggest a strong association between IgG antiguanosine antibodies and major manifestations of procainamide-induced systemic lupus erythematosus.

Antibodies to DNA.

Antibodies that recognize specific conformational variations of DNA structure provide sensitive reagents for testing the extent to which such conformational heterogeneity occurs in nature. A most dramatic recent example has been the development and application of antibodies to left-handed Z-DNA. They provided the first identification of Z-DNA in fixed nuclei and chromosomes, and of DNA sequences that form Z-DNA under the influence of supercoiling. Antibodies have also been induced by chemically modified DNA and by synthetic polydeoxyribonucleotides that differ from the average B-DNA structure. These antibodies recognize only the features that differ from native DNA. In most experiments, native DNA itself is not immunogenic. Antibodies that do react with native DNA occur in sera of patients with autoimmune disease, but even monoclonal anti-DNA autoantibodies usually react with other polynucleotides as well. Anti-DNA antibodies, especially those of monoclonal origin, provide a model for the study of protein-nucleic acid recognition.

Mechanism of coupling periodate-oxidized nucleosides to proteins.

This paper describes a mechanism of coupling periodate-oxidized nucleosides to proteins. Each of the dialdehyde groups of a periodate-oxidized nucleoside is shown to couple to lysine residues on different protein molecules through Schiff bases, thereby cross-linking different protein molecules, forming a polymer. This is in contrast to the previous model in which nucleosides were suggested to couple to proteins through a morpholine structure. The cross-linked structure of the nucleoside-antigen, significantly different when compared to the native protein, may affect the specificity and the efficiency of antibody production.

The fine specificity of IgG antiguanosine antibodies in systemic lupus erythematosus.

The antigen specificity, isotype, and subclass of antinuclear antibodies may be related to their pathogenicity in systemic lupus erythematosus (SLE). Our laboratory found that IgG antibodies that bound the nucleoside, guanosine, occurred frequently in SLE patients. In contrast, sera from healthy subjects contained IgM but not IgG antiguanosine antibodies. The present studies were designed to characterized the fine specificity of IgG antiguanosine antibodies in SLE and compare them with IgM antiguanosine antibodies in normal sera. Serum antinuclear antibodies from six healthy subjects and six SLE patients were isolated by affinity binding to guanosine and measured by an enzyme-linked immunosorbent assay (ELISA). IgM in normal sera, and both IgM and IgG in SLE sera bound guanosine. IgM antiguanosine antibodies in normal sera were polyspecific and bound other nucleosides and 1-methylguanosine but not denatured DNA (ssDNA). In contrast, IgG antiguanosine antibodies from the SLE patients bound guanosine and ssDNA but not other nucleosides or 1-methylguanosine. SLE IgM antiguanosine antibodies had the same fine specificity and bound guanosine and ssDNA but not any of the other nucleosides. These results suggest that SLE IgG and IgM antiguanosine antibodies have fine specificity in contrast to the polyspecific IgM antibodies in normal sera. In addition, subclass analysis indicated that all SLE patients had either IgG1 or IgG3 subclass of antiguanosine antibodies that bind complement. Characterizing the isotype, subclass, and fine antigen specificity of antiguanosine antibodies should assist in evaluating their potential pathogenicity in SLE.

Immunological detection of lesions in DNA.

The purpose of this paper is to show that the antibodies to nucleic acids, to nucleosides or to DNA damaged by a physical or a chemical agent, are useful tools in the study of DNA damage and repair. The results obtained with antibodies to nucleosides, antibodies to nucleosides and DNA modified by chemical carcinogens emphasize the potential of immunological methods in three main areas, a) the sensitive detection and quantitation of adducts; b) the visualization of adducts in tissues, individual cells, and along the DNA double helix; c) the study of conformational changes of DNA induced by adducts.

Antibody-nucleic acid complexes. Antigenic domains within nucleosides as defined by solid-phase immunoassay.

The usefulness of solid-phase immunoassays for the characterization of anti-nucleoside antibodies was investigated. Antibodies specific for guanosine (G), 7-methyl-guanosine (m7G), and cytidine (C) were obtained from the serum of rabbits immunized with nucleoside-KLH (keyhole limpet hemocyanin) conjugates. Solid-phase assays consisted of measuring the ability of these antibodies to be retained by microtiter wells containing immobilized nucleoside-BSA (bovine serum albumin) conjugates. Nucleosides employed as haptens included adenosine (A), N6-methyl-A (m6A), guanosine (G), N2,N2-dimethyl-G (m22G), 1-methyl-G (m1G), O6-methyl-G (m6G), 7-methyl-G (m7G), cytidine (C), 5-methyl-C (m5C), uridine (U), and ribothymidine (T). Spectral analysis of these conjugates revealed that 15-20 nucleosides were coupled to each BSA molecule. Quantitative information regarding the various reactions associated with these assays was obtained by employing antigen and antibody (IgG) preparations radiochemically labeled via reductive methylation using NaB3H4 and formaldehyde (specific activities 0.6-2.1 X 10(6) cpm/micrograms). Data obtained with 3H-labeled antigens indicated that the adsorption of all nucleoside-BSA conjugates was uniform and irreversible with respect to the assay conditions used. Assays designed to measure antibody binding in the presence of excess antigen revealed that (i) nonspecific binding to immobilized BSA was negligible, (ii) as little as 0.5 ng of bound antibody could be detected, (iii) antibody retention was directly proportional to antibody concentration, and (iv) each anti-nucleoside antibody cross-reacted to a considerable extent with nonhomologous haptens.(ABSTRACT TRUNCATED AT 250 WORDS)