991. Blockade of the PD-1/PD-L1 Immune Checkpoint Pathway Improves Mortality, Infection Severity, and Fungal Clearance in an Immunosuppressed Murine Model of Invasive Pulmonary Mucormycosis Ibrexafungerp in Patients with Oropharyngeal and from an Interim Analysis of a Phase 3 Open-label

Background. Emerging experimental evidence suggests that immune checkpoint inhibitors (ICIs) enhance antifungal immunity. In addition, there is anecdotal evidence of potential benefit of adjunct PD-1 pathway blockade in patients with intractable mucormycosis. However, proof-of-concept data in animal models are lacking. Therefore, we compared the efficacy of PD-1 and PD-L1 inhibition in an immunosuppressed murine model of invasive pulmonary mucormycosis (IPM). Methods. Female 8-9-week-old BALB/c mice were immunosuppressed with cyclophosphamide (150 mg/kg on days -4 and -1, 100 mg/kg on day +3) and corti-sone acetate (300 mg/kg on day -1) and infected intranasally with 50,000 Rhizopus arrhizus spores (clinical isolate Ra-749, day 0). On days 0, +2, +4, and +6, mice received intraperitoneal injections of 250 µg/kg PD-1 or PD-L1 blocking antibodies versus (vs.) 250 µg/kg of the corresponding isotype antibodies (all antibodies from Leinco Technologies). Survival was monitored for 7 days post-infection. Infection severity was scored using the murine sepsis score (MSS, 0 = healthy to 3 = mori-bund). Fungal burden in lung tissue was determined by an 18S quantitative PCR assay on day +7 or upon death. 20 mice per treatment were assessed in 2 independent experiments. Results. Control mice with IPM receiving either of the unspecific isotype anti- bodies developed severe infection (median MSS on day 2.5-3.0) and had a high 7-day mortality (50-55%). Compared to the corresponding isotype control, PD-L1 inhibition provided a strong therapeutic benefit, significantly improving morbidity (median MSS = 1.0 vs. 2.5, p = 0.002), 7-day mortality (15% vs. 50%, p = 0.02), and fungal (3.6k vs. spore equivalents/lung, p < 0.001). In contrast, modestly yet non-significantly reduced infection severity (me- dian MSS 2.1 vs. 3.0, p = 0.48), 7-day mortality (35% vs, 55%, p = 0.12), and fungal (5.6k vs. 40.7k spore equivalents/lung, p = 0.09) compared to isotype control. Conclusion. Even without concomitant antifungals, blockade of PD-L1 and to a lesser extent of PD-1 improved mortality, infection severity, and fungal clearance in immunosuppressed mice with IPM. Immune phenotyping studies are in progress to better understand the protective antifungal activity of ICIs in IPM. metagenomic sequencing classify sequences generate map. a robust methodology to acquire viral genomic data, microbiome com- position, co-infection information, and the transcriptional status of the immune response in a single workflow. This sequencing-based approach can be available on the first day of the next viral outbreak and should be considered as a first-line test for novel zoonotic virus detection. Bacterial species composition of patient stool samples before and after CRISPRclean depletion.


FURI Study Group
Session: P-55. Medical Mycology Background. Candida albicans is the predominant organism causing esophageal candidiasis (EC) and oropharyngel candidiasis (OPC). These infections may arise from subjects colonized with Candida who are predisposed due to illness, debility or a local reduction in host resistance to an overgrowth of their own indigenous flora. Patients with mucocutaneous Candida infections can be treated in the outpatient setting, yet there are limited oral treatment options available for patients who are unresponsive to or who are intolerant to currently available antifungals. Oral ibrexafungerp is an investigational broad-spectrum glucan synthase inhibitor antifungal with activity against Candida species, including azole-and echinocandin-resistant strains. A Phase 3 open-label, single-arm study of ibrexafungerp (FURI; NCT03059992) is ongoing for the treatment of patients intolerant of or with fungal disease refractory to standard antifungal therapy. Table 1. FURI Outcomes in OPC and EC Methods. FURI subjects were eligible for enrollment if they had proven or probable severe mucocutaneous candidiasis, invasive candidiasis, invasive aspergillosis, or other fungal diseases with evidence of treatment failure, intolerance, or toxicity related to a currently approved standard-of-care antifungal treatment or if they were unable to receive an approved oral antifungal option (e.g., susceptibility of the organism) and a continued IV antifungal therapy was clinically undesirable or unfeasible.
Results. An independent Data Review Committee (DRC) provided an assessment of treatment response for 74 subjects enrolled in the FURI study from 22 centers in US, UK and EU treated with ibrexafungerp for mucocutaneous or invasive fungal infections from 2016-2020. A total 32 subjects (43.2%) had mucocutaneous candidiasis and 24 subjects were diagnosed with OPC or EC. The percent of patients who were determined to have a complete response (CR) or partial response (PR) was 62.5%, stable disease (SD), 20.8%, and progression of disease, 16.7%. Table 1 shows outcomes by EC and OPC as determined by the DRC.
Conclusion. Analysis of 24 EC and OPC patients from the FURI study indicates that oral ibrexafungerp provides a favorable therapeutic response in patients with challenging mucocutaneous fungal disease and limited treatment options.
Disclosures. Oliver Cornely, Prof., The COVID-19 pandemic has brought awareness to the dangers of emerging pathogens to global human health and welfare. Sensitivity and flexibility are important features for methods used to detect emerging pathogens. While PCR testing is rapid and sensitive, a significant advantage next generation sequencing (NGS) approaches have over PCR-based analyses is the ability to detect previously undiscovered pathogens while also providing genomic information that can detect SARS-CoV-2 genome sequence, identify source of co-infection, and the host transcriptional response in a single workflow. The critical component enabling this approach is Jumpcode CRISPRclean technology which removes abundant human and bacterial ribosomal RNA sequences from NGS libraries.

CRISPRclean workflow easily integrates into next generation sequencing projects
Schematic of the Jumpcode CRISPRclean protocol Methods. CRISPRclean was applied to contrived infected tissue samples including human lung RNA spiked with serially diluted amounts of SARS-CoV-2 RNA and bacterial RNA. NEB RNA libraries were prepared and treated with CRISPRclean protocol, then sequenced on Illumina instruments. Data analysis was performed using Jumpcode proprietary software to measure alignment and depletion rates, the Silva database for rRNA read alignment, and Kraken2 and CosmosID pipelines for k-mer based metagenomic investigation. Fold enrichment of SARS-CoV-2 reads after CRISPRclean depletion of libraries prepared from contrived samples.
CRISPRclean treatment of the fully contrived samples increases the fraction of reads that map to the SARS-CoV-2 genome by an average of ~10-fold Results. CRISPRclean treatment of the contrived samples increases ~10 fold of reads that map to the SARS-CoV-2 genome. For the 60 viral copies of SARS-CoV-2 sample, the number of reads mapping to the SARSCoV-2 genome increases from ~10,000 reads to ~70,000 reads. A similar increase in reads occurs for S. aureus. The percentage of SARS-CoV-2 genome covered at 1X and 10X also increases. Similar results were achieved even after downsampling the datasets to 5M reads. There is a ~4-fold increase in bacterial species detection in these stool samples after CRISPRclean treatment. Percentage of SARS-CoV-2 genome covered at 1X and 10X increases as a result of rRNA depletion.
Coverage of the SARS-CoV-2 genome at 50 million reads. Number of reads aligning to the S. aureus and SARS-CoV-2 genomes increases after CRISPRclean depletion.
For the sample containing 0.0001% SARS-CoV-2, (60 viral copies), the number of reads mapping to the SARS-CoV-2 genome increases from ~10,000 reads to ~70,000 reads.
CosmosID Shotgun Metagenomics Analysis heat map showing read alignments to viral genomes. The yellow color indicates high read counts. The CosmosID shotgun metagenomic analysis software was used to analyze the sequencing data, classify the sequences and generate the heat map.
Conclusion. Metatranscriptomics powered by CRISPR-mediated rRNA depletion offers a robust methodology to acquire viral genomic data, microbiome composition, co-infection information, and the transcriptional status of the host immune response in a single workflow. This sequencing-based approach can be available on the first day of the next viral outbreak and should be considered as a first-line test for novel zoonotic virus detection. Bacterial species composition of patient stool samples before and after CRISPRclean depletion.