The effect of poly(ADP‐ribose) synthesis on chromatin structure was investigated by velocity sedimentation and electron microscopy. We demonstrate that locally relaxed regions can be generated within polynucleosome chains by the activity of their intrinsic poly(ADP‐ribose)polymerase. This relaxation phenomenon is also shown to be NAD dependent and to be correlated with the formation of hyper(ADP‐ribosyl)ated forms of histone H1. Evidence is also presented which suggests that hyper(ADP‐ribosyl)ated histone H1 is neither released from the relaxed chromatin, nor does it seem to participate in polynucleosomal aggregation.

The pattern of nucleosomal histones poly(ADP-ribosy1)ation is changed under conditions which affect the poly(ADP-ribosy1)ation state of the enzyme. At low NAD concentrations the enzyme can poly(ADP-ribosy1)ate histones H1 and H1 O, H2A, A2A, and H2B. However at NAD concentrations above 10 yM the enzyme preferentially poly(ADP-ribosy1)ates histone H1 to a hyper ADP-ribosylated form. Furthermore we have observed hyper ADP-ribosylation of histone H2B at NAD concentrations of 10 yM suggesting that histone H2B can undergo the same type of ADP-ribosylation pattern as histone H1. Also at higher NAD concentrations an elongation of the polymer attached to the enzyme and other nuclear proteins takes place.There is growing evidence that poly(ADP-ribosy1)ation of nucleosomal proteins, which is a post-translational modification, might be related to the alteration of the chromatin structure during the cellular process of DNA repair and replication [l -31. The reaction is catalyzed by the nuclear enzyme poly(ADP4bose) polymerase [4 -61 which can act not only as a catalyst but also as the acceptor of the product of its own activity [7-91. Amongst the histone proteins, it has been shown that histones HI, H2B, A24 and to a lesser extent histones H3 and H2A could be poly(ADP-ribosy1)ated in nuclei as well as in nucleosomes [lo-121. Histone HI has been described to be the major histone acceptor in trout testis nuclei [13] and pancreatic nuclei and nucleosomes [14, 151 at NAD concentrations above 100 pM; whereas in rat liver and HeLa cells histones H1 and H2B have been shown to be the major histone protein acceptors of poly(ADP-ribose) [12,16].The biological role of the polymer has been ascribed to different nuclear events such as: DNA replication [17], DNA repair [18, 191, cell growth and differentiation [20, 211. Furthermore it has been shown that there is an increase in the poly(ADP-ribosy1)ation of histone HI during DNA repair in permeabilized cells [22]. Also studies in vivo have shown increased poly(ADP-ribosy1)ation of histone H1 and core histones [23] during DNA repair. Cell cycle studies have indicated increased poly(ADP-ribosy1)ation of histone H1 during S phase [24]. It has been suggested recently by Poirier et al. [25] that poly(ADP-ribosy1)ation of histone H1 could alter the chromatin structure and be one of the mechanisms by which such nuclear events could be facilitated [25]. Indeed, they observed an opening of the chromatin structure, that is a relaxation, following the poly(ADP-ribosy1)ation of histone H1 both upon the addition of exogenous poly(ADP-ribose) polymerase [25] or by the intrinsic enzymatic activity of the nucleosomes [26].Abbreviations. Poly(ADP-ribose), polymer of adenosine 5'-diphosphate ribose; LDS, lithium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.Enzyme. Poly(ADP-ribose)polymerase (EC 2.4.99. -).In order to have a better understanding of the interaction between poly(ADP-ribose) polymerase and nucleosomal proteins during poly(ADP-ribosyl)ation, we have performed acceptor studies und...

We have studied the kinetics of relaxation of poly(ADP-ribosyl)ated polynucleosomes produced by endogenous enzyme activity by comparing the generation of hyper(ADP-ribosyl)ated histone H1 and its effect on the chromatin structure as revealed by electron microscopy. A correlation can be established between the appearance of histone H1 modified forms and the localized relaxation of the chromatin. We have also noticed, in parallel, that poly(ADP-ribosyl)ated chromatin showed increased solubility in the presence of Mg2+ and 0.2 M NaCl. Electron microscopic examination of the solubilized chromatin produced by poly(ADP-ribosyl)ation shows polynucleosomes exhibiting more relaxed conformation, whereas an increasing amount of hyper(ADP-ribosyl)ated histone H1 is found in the pellet, as shown by acid-urea-polyacrylamide electrophoretic separation of histone extracts.

The rapid spread of SARS-CoV-2 continues to impact humanity on a global scale with rising total morbidity and mortality. Despite the development of several effective vaccines, new products are needed to supply ongoing demand and to fight variants. We report herein a pre-specified interim analysis of the phase 2 portion of a Phase 2/3, randomized, placebo-controlled trial of a coronavirus virus-like particle (CoVLP) vaccine candidate, produced in plants that displays the SARS-CoV-2 spike glycoprotein, adjuvanted with AS03 (NCT04636697). A total of 753 participants were recruited between 25th November 2020 and 24th March 2021 into three groups: Healthy Adults (18–64 years: N = 306), Older Adults (≥65 years: N = 282) and Adults with Comorbidities (≥18 years: N = 165) and randomized 5:1 to receive two intramuscular doses of either vaccine (3.75 µg CoVLP/dose+AS03) or placebo, 21 days apart. This report presents safety, tolerability and immunogenicity data up to 6 months post-vaccination. The immune outcomes presented include neutralizing antibody (NAb) titres as measured by pseudovirion assay at days 21 and 42 as well as neutralizing antibody cross-reactivity to several variants of concern (VOCs): Alpha, Beta, Gamma, Delta, and Omicron (BA.1), up to 201 days post-immunization. Cellular (IFN-γ and IL-4 ELISpot) response data in day 21 and 42 peripheral blood are also presented. In this study, CoVLP+AS03 was well-tolerated and adverse events (AE) after each dose were generally mild to moderate and transient. Solicited AEs in Older Adults and Adults with Comorbidities were generally less frequent than in Healthy Adults and the reactogenicity was higher after the second dose. CoVLP+AS03 induced seroconversion in >35% of participants in each group after the first dose and in ~98% of participants, 21 days after the second dose. In all cohorts, 21-days after the second dose, NAb levels in sera against the vaccine strain were ~10-times those in a panel of convalescent sera. Cross-reactivity to Alpha, Beta and Delta variants was generally retained to day 201 (>80%) while cross-reactivity to the Gamma variant was reduced but still substantial at day 201 (73%). Cross-reactivity to the Omicron variant fell from 72% at day 42 to 20% at day 201. Almost all participants in all groups (>88%) had detectable cellular responses (IFN-γ, IL-4 or both) at 21 days after the second dose. A Th1-biased response was most evident after the first dose and was still present after the second dose. These data demonstrated that CoVLP+AS03 is well-tolerated and highly immunogenic, generating a durable (at least 6 months) immune response against different VOCs, in adults ≥18 years of age, with and without comorbidities.

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