During meiotic prophase, a structure called the synaptonemal complex (SC) assembles at the interface between aligned pairs of homologous chromosomes, and crossover recombination events occur between their DNA molecules. Here we investigate the inter-relationships between these two hallmark features of the meiotic program in the nematode C. elegans, revealing dynamic properties of the SC that are modulated by recombination. We demonstrate that the SC incorporates new subunits and switches from a more highly dynamic/labile state to a more stable state as germ cells progress through the pachytene stage of meiotic prophase. We further show that the more dynamic state of the SC is prolonged in mutants where meiotic recombination is impaired. Moreover, in meiotic mutants where recombination intermediates are present in limiting numbers, SC central region subunits become preferentially stabilized on the subset of chromosome pairs that harbor a site where pro-crossover factors COSA-1 and MutSγ are concentrated. Polo-like kinase PLK-2 becomes preferentially localized to the SCs of chromosome pairs harboring recombination sites prior to the enrichment of SC central region proteins on such chromosomes, and PLK-2 is required for this enrichment to occur. Further, late pachytene nuclei in a plk-2 mutant exhibit the more highly dynamic SC state. Together our data demonstrate that crossover recombination events elicit chromosome-autonomous stabilizing effects on the SC and implicate PLK-2 in this process. We discuss how this recombination-triggered modulation of SC state might contribute to regulatory mechanisms that operate during meiosis to ensure the formation of crossovers while at the same time limiting their numbers.

GS-9688 (selgantolimod) is an oral selective small molecule agonist of toll-like receptor 8 (TLR8) in clinical development for the treatment of chronic hepatitis B (CHB). In this study, we evaluated the antiviral efficacy of GS-9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus (HBV). WHV-infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS-9688 or 3 mg/kg GS-9688. Vehicle and 1 mg/kg GS-9688 had no antiviral effect, whereas 3 mg/kg GS-9688 induced a >5 log 10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS-9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti-WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS-9688 was confirmed in a second woodchuck study. The antiviral response to GS-9688 did not correlate with systemic GS-9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells (PBMC) to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells (T FH) and various other immune cell subsets may play a role in the antiviral response to GS-9688. Conclusion: Finite, short-duration treatment with a clinically relevant dose of GS-9688 is well tolerated and can induce a sustained antiviral response in WHV-infected woodchucks. The identification of a baseline intrahepatic transcriptional signature associated with response to GS-9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect. Approximately 260 million individuals are chronically infected with hepatitis B virus (HBV), and over half a million people are estimated to die each year due to liver diseases associated with chronic hepatitis B (CHB), such as cirrhosis and hepatocellular carcinoma (HCC). Immunological control of CHB ("functional cure") is defined as sustained loss of HBV surface antigen (HBsAg) off treatment, with or without seroconversion to anti-HBs antibody. Several nucleos(t)ide analogs, as well as interferon-alpha (IFN-α), are approved for the treatment of CHB. These therapies reduce viremia and

21During meiotic prophase, a structure called the synaptonemal complex (SC) assembles at the 22 interface between aligned pairs of homologous chromosomes, and crossover recombination 23 events occur between their DNA molecules. Here we investigate the inter-relationships between 24 these two hallmark features of the meiotic program in the nematode C. elegans, revealing 25 dynamic properties of the SC that are modulated by recombination. We demonstrate that the 26 SC incorporates new subunits and switches from a more highly dynamic/labile state to a more 27 stable state as germ cells progress through the pachytene stage of meiotic prophase. We 28 further show that the more dynamic state of the SC is prolonged in mutants where meiotic 29 recombination is impaired. Moreover, in meiotic mutants where recombination intermediates are 30 present in limiting numbers, SC central region subunits become preferentially stabilized on the 31 subset of chromosome pairs that harbor a site where pro-crossover factors COSA-1 and MutSγ 32 are concentrated. Polo-like kinase PLK-2 becomes preferentially localized to the SCs of 33 chromosome pairs harboring recombination sites prior to the enrichment of SC central region 34 proteins on such chromosomes, and PLK-2 is required for this enrichment to occur. Further, 35 late pachytene nuclei in a plk-2 mutant exhibit the more highly dynamic SC state. Together our 36 data demonstrate that crossover recombination events elicit chromosome-autonomous 37 stabilizing effects on the SC and implicate PLK-2 in this process. We discuss how this 38 recombination-triggered modulation of SC state might contribute to regulatory mechanisms that 39 operate during meiosis to ensure the formation of crossovers while at the same time limiting 40 their numbers. 41. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/110064 doi: bioRxiv preprint first posted online 3 Author Summary 42Reliable chromosome inheritance during sexual reproduction depends on the formation of 43 temporary connections between homologous chromosomes that enable them to segregate 44 toward opposite spindle poles at the meiosis I division. These connections are established 45 during an extended meiotic prophase characterized by two prominent features: a highly-46 ordered structure called the synaptonemal complex (SC) that assembles at the interface 47 between aligned pairs of chromosomes, and crossover recombination events between their 48 DNA molecules that are completed in the context of the SC. In the current work, we investigate 49 the inter-relationships between these two hallmark features of the meiotic program in the 50 nematode C. elegans. Our work reveals the C. elegans SC as a much more dynamic structure 51 than is suggested by its highly-ordered appearance in EM images, and further demonstrates 52 that the SC switches from a more highly dynamic/labile state to a more stable state ...

BaCKgRoUND aND aIMS: GS-9688 (selgantolimod) is a toll-like receptor 8 agonist in clinical development for the treatment of chronic hepatitis B (CHB). Antiviral activity of GS-9688 has previously been evaluated in vitro in HBVinfected hepatocytes and in vivo in the woodchuck model of CHB. Here we evaluated the potential of GS-9688 to boost responses contributing to viral control and to modulate regulatory mediators.appRoaCH aND ReSUltS: We characterized the effect of GS-9688 on immune cell subsets in vitro in peripheral blood mononuclear cells of healthy controls and patients with CHB. GS-9688 activated dendritic cells and mononuclear phagocytes to produce IL-12 and other immunomodulatory mediators, inducing a comparable cytokine profile in healthy controls and patients with CHB. GS-9688 increased the frequency of activated natural killer (NK) cells, mucosalassociated invariant T cells, CD4 + follicular helper T cells, and, in about 50% of patients, HBV-specific CD8 + T cells expressing interferonγ. Moreover, in vitro stimulation with GS-9688 induced NK-cell expression of interferonγ and TNFα, and promoted hepatocyte lysis. We also assessed whether GS-9688 inhibited immunosuppressive cell subsets that might enhance antiviral efficacy. Stimulation with GS-9688 reduced the frequency of CD4 + regulatory T cells and monocytic myeloid-derived suppressor cells (MDSCs). Residual MDSCs expressed higher levels of negative immune regulators, galectin-9 and programmed death-ligand 1. Conversely, GS-9688 induced an expansion of immunoregulatory TNFrelated apoptosis-inducing ligand + NK cells and degranulation of arginase-I + polymorphonuclear MDSCs.CoNClUSIoNS: GS-9688 induces cytokines in human peripheral blood mononuclear cells that are able to activate antiviral effector function by multiple immune mediators (HBVspecific CD8 + T cells, CD4 + follicular helper T cells, NK cells, and mucosal-associated invariant T cells). Although reducing the frequency of some immunoregulatory subsets, it enhances the immunosuppressive potential of others, highlighting potential biomarkers and immunotherapeutic targets to optimize the antiviral efficacy of GS-9688. (Hepatology 2021;0:1-17). C hronic hepatitis B (CHB) remains a global health concern with an estimated 260 million people infected worldwide. CHB

Meiotic recombination is initiated by the programmed induction of double-strand DNA breaks (DSBs), lesions that pose a potential threat to the genome. A subset of the DSBs induced during meiotic prophase become designated to be repaired by a pathway that specifically yields interhomolog crossovers (COs), which mature into chiasmata that temporarily connect the homologs to ensure their proper segregation at meiosis I. The remaining DSBs must be repaired by other mechanisms to restore genomic integrity prior to the meiotic divisions. Here we show that HIM-6, the Caenorhabditis elegans ortholog of the RecQ family DNA helicase BLM, functions in both of these processes. We show that him-6 mutants are competent to load the MutSg complex at multiple potential CO sites, to generate intermediates that fulfill the requirements of monitoring mechanisms that enable meiotic progression, and to accomplish and robustly regulate CO designation. However, recombination events at a subset of CO-designated sites fail to mature into COs and chiasmata, indicating a pro-CO role for HIM-6/BLM that manifests itself late in the CO pathway. Moreover, we find that in addition to promoting COs, HIM-6 plays a role in eliminating and/or preventing the formation of persistent MutSg-independent associations between homologous chromosomes. We propose that HIM-6/BLM enforces biased outcomes of recombination events to ensure that both (a) CO-designated recombination intermediates are reliably resolved as COs and (b) other recombination intermediates reliably mature into noncrossovers in a timely manner.