Myeloid dendritic cells (DCs) have the innate capacity to sense pathogens and orchestrate immune responses. However, DCs do not mount efficient immune responses to HIV-1, primarily due to restriction of virus reverse transcription, which prevents accumulation of viral cDNA and limits its detection through the cGAS-STING pathway. By allowing reverse transcription to proceed, we find that DCs detect HIV-1 in distinct phases, before and after virus integration. Blocking integration suppresses, but does not abolish, activation of the transcription factor IRF3, downstream interferon (IFN) responses, and DC maturation. Consistent with two stages of detection, HIV-1 "primes" chromatin accessibility of innate immune genes before and after integration. Once primed, robust IFN responses can be unmasked by agonists of the innate adaptor protein, MyD88, through a process that requires cGAS, STING, IRF3, and nuclear factor κB. Thus, HIV-1 replication increases material available for sensing, and discrete inflammatory inputs tune cGAS signaling to drive DC maturation.

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Highlights d Network inference reveals a transcriptional map of the innate immune response to HIV d Integrating cell-specific chromatin accessibility data improves network inference d Immune responses coincide with network enrichment of IRF, STAT, and NF-kB factors d RARA and PRDM1 are validated as additional regulators of the innate response to HIV

Cyclospora cayetanensis is the causative agent of cyclosporiasis, an emerging infectious disease. We present a new method for the purification of C. cayetanensis oocysts from feces using a modified detachment solution and Renocal-sucrose gradient sedimentation. This method yields oocysts free from adherent fecal debris and amenable to processing using flow cytometry.

Cyclospora cayetanensis, a protozoan of emerging concern, causes self-limiting gastroenteritis in immune-competent hosts. It has been established that sequence variability exists in the first internal transcribed spacer region (ITS-1) of the ribosomal DNA operon from collections of oocysts obtained from individual or pooled fecal samples. To determine if single oocysts also exhibited ITS-1 sequence variability, DNA was extracted from individually flow-cytometry-counted oocysts. We determined that ITS-1 sequence variability exists at an individual-genome level for C. cayetanensis and approached or exceeded the variability exhibited among oocyst collections. ITS-1 variability, at the genome level, reduces this region's utility for inferring relationships between strains.

The departure of many pharmaceutical companies from antibiotic research starting in the 1980s resulted in the absence of new antibiotics to combat the current crisis of increasing microbial resistance to currently available antibiotics. In an effort to address the increasing need for novel antibiotics, AMRI began a screening campaign to identify potent compounds from our natural product library. Natural products, particularly those produced by microbial fermentation, were the direct source or inspiration for almost all antibiotics used today and remain the richest source for new antibacterial compound series. AMRI's extensive library consisting of over 280 000 samples was screened for activity against a multi-drug resistant strain of Staphylococcus aureus (ATCC 43 300, Rockville, MD, USA). The hits arising out of this assay were then tested against the human hepatocellular carcinoma cell line HepG2 to filter out those samples where activity was the result of general cytotoxicity and determine an in vitro therapeutic index (in vitro TI). Samples with a high in vitro TI were selected for fractionation and dereplication. The resulting subset of samples possessing selectivity for the bacterial target were then fractionated on an HPLC system employing UV, ELSD (evaporative light scattering) and MS detectors. The eluted fractions were collected into 96-well microtiter plates and submitted for bioassay. LC/MS data for the active fractions generated UV spectra and molecular weights, which were used to search internal and external databases. 1 The analysis of the extract from strain 4731, which exhibited excellent activity against several Gram-positive organisms, resulted in the discovery of a new anthraquinone, which was closely related to the known antibiotic ericamycin. 2,3 Here we report the isolation, structure elucidation and biological activities of 1.The Actinoplanes sp. strain 4731 was isolated from a soil sample collected from grassland near Nanton, in Alberta, Canada. The culture was isolated by a previously described capillary chemotaxis technique, 4 using 0.18% mannitol as a chemo-attractant, spread-plating on water-yeast extract agar plates 5 and incubating in the dark at 28 1C for 10 days. All colonies visible under a dissecting scope were transferred to and purified on starch casein agar plates. Pure cultures were macerated and stored in a 10% glycerol/5% lactose solution at À80 1C. Before fermentation, the culture was streaked from a cryovial onto starch casein agar to verify purity. Fresh culture macerate was prepared from these agar plates after 14 days and used to inoculate the fermentation. Strain 4731 was identified by sequencing of the 16S rRNA gene. Genomic DNA was isolated from a culture grown in tryptic soy broth for 7 days at 28 1C by a phenol:chloroform extraction method. 6 The 16S rRNA gene was amplified using Taq polymerase (Promega, Madison, WI, USA) and the following two primer pairs: 27F, 5 0 -AGA GTTTGATCMTGGCTCAG-3 0 ; 1115R, 5 0 -AGGGTTGCGCTCGTTG-3 0 ; and 339F, 5 0 -CTCCTACGGGAGGCAGCA...

BackgroundSGN-B7H4V is a novel, investigational vedotin antibody drug conjugate (ADC) directed to B7-H4, a member of the B7 family of immune checkpoint ligands. B7-H4 expression is elevated on a variety of solid tumors including breast, ovarian, and endometrial tumors.1 SGN-B7H4V is composed of a fully human IgG1 anti-B7-H4 monoclonal antibody (mAb) conjugated to the microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable peptide linker. SGN-B7H4V is designed to bind and internalize the immune checkpoint ligand B7-H4/ADC complex from the surface of malignant cells and release the cytotoxic payload MMAE. This ”vedotin” drug linker system has been clinically validated by multiple ADC programs, including brentuximab vedotin, enfortumab vedotin, and polatuzumab vedotin.2–4 Here, we characterize the target antigen B7-H4 and evaluate SGN-B7H4V activity in preclinical models.MethodsB7-H4 expression was characterized by RNA expression and immunohistochemistry across multiple solid tumor types. The ability of SGN-B7H4V to kill B7-H4-expressing tumor cells in vitro and in vivo in a variety of xenograft tumor models was also evaluated. Finally, the tolerability of SGN-B7H4V was assessed in rodent and non-human primate toxicology studies.ResultsImmunohistochemistry confirmed expression of B7-H4 across multiple solid tumor types, including ovarian and breast tumors. In vitro, upon binding to SGN-B7H4V, the immune checkpoint ligand B7-H4 was rapidly internalized and delivered the cytotoxic payload MMAE. Moreover, SGN-B7H4V killed B7-H4-expressing tumor cells in vitro by MMAE-mediated cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). In vivo, SGN-B7H4V demonstrated strong anti-tumor activity in multiple xenograft models, including ovarian and breast cancer models. Activity was observed in models with both uniformly high and heterogeneous expression of B7-H4, consistent with robust bystander activity of vedotin ADCs. Finally, SGN-B7H4V was tolerated in both rat and non-human primate (NHP) toxicology studies at doses consistent with approved vedotin ADCs.ConclusionsB7-H4 is a promising ADC target expressed by several solid tumor types. SGN-B7H4V demonstrates robust anti-tumor activity in preclinical models through multiple potential mechanisms and is tolerated in rat and NHP toxicity studies. Altogether, these data support further evaluation of SGN-B7H4V in a planned, first-in-human phase 1 clinical study.AcknowledgementsWe would like to thank Kellie Spahr for conjugation support and Martha Anderson for in vivo biology support.ReferencesLeong SR, Liang WC, Wu Y, Crocker L, Cheng E, Sampath D, et al. An anti-B7-H4 antibody-drug conjugate for the treatment of breast cancer. Mol Pharm 2015;12(6):1717–29. Epub 2015/04/09. doi: 10.1021/mp5007745. PubMed PMID: 25853436.Rosenberg JE, O’Donnell PH, Balar AV, McGregor BA, Heath EI, Yu EY, et al. Pivotal trial of enfortumab vedotin in urothelial carcinoma after platinum and anti-programmed death 1/programmed death ligand 1 therapy. J Clin Oncol 2019;37(29):2592–600. Epub 2019/07/30. doi: 10.1200/JCO.19.01140. PubMed PMID: 31356140; PubMed Central PMCID: PMC.Senter PD, Sievers EL. The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat Biotechnol 2012;30(7):631–7. Epub 2012/07/12. doi: 10.1038/nbt.2289. PubMed PMID: 22781692.Tilly H, Morschhauser F, Bartlett NL, Mehta A, Salles G, Haioun C, et al. Polatuzumab vedotin in combination with immunochemotherapy in patients with previously untreated diffuse large B-cell lymphoma: an open-label, non-randomised, phase 1b-2 study. Lancet Oncol 2019;20(7):998–1010. Epub 2019/05/19. doi: 10.1016/S1470-2045(19)30091–9. PubMed PMID: 31101489.Ethics ApprovalAll animal studies were conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee at Seagen or the external testing facility that conducted the studies.

Transcriptional programming of the innate immune response is pivotal for host protection. However, the transcriptional mechanisms that link pathogen sensing with innate activation remain poorly understood. During infection with HIV-1, human dendritic cells (DCs) can detect the virus through an innate sensing pathway leading to antiviral type I interferon and DC maturation. Here, we have developed an iterative experimental and computational approach to map the innate response circuitry during HIV-1 infection. By integrating genome-wide chromatin accessibility with expression kinetics, we have inferred a gene regulatory network that links 542 transcription factors (TFs) with 21,862 target genes. Through genetic perturbation and drug treatments we identify PRDM1 and RARA as essential regulators of the interferon response and DC maturation, respectively. Our work provides a resource for interrogation of regulators of HIV replication and innate immunity, highlighting the complexity and cooperativity in the regulatory circuit controlling the DC response to HIV-1 infection.