CTR056040 Supplier Grant Application UA_Daines.pdf

Table of Contents

Attachment B: Application Packet General Information W-9 Review Purposes Project Abstract Detailed Project Description ........................................................................................................ 6-14

Project Activities ............................................................................................................. 6-12 Project Resources ........................................................................................................ 12-13 References

Biographical Sketches:

Michael O. Daines, MD ............................................................................................... 15-19 Pawel R. Kiela, DVM, PhD ........................................................................................... 20-24 Sydney Rice, PhD ........................................................................................................ 25-27

Budget Information: ................................................................................................................... 28-33 Budget Year 1 Budget Year 2 Budget Year 3 Cumulative Budget Justification ...................................................................................................... 32-33

Certifications and Assurances:

Drug-Free Workplace Disclosure of Lobbying Activities Debarment and Suspension ........................................................................................ 36-37 Single Audit Certification

Attachment C: Grant Offer and Acceptance Attachment D: Notices, Correspondence, and Payments Attachment E: Designation of Confidential Trade Secret and Proprietary Information ......................... 41-42 Attachment F: Participation of Boycott of Israel Form

Table of ContentsMichael Daines, ADHS BPM000334 (AZ IG) 1 of 43

General Information

Information about the Applicant organization or individual and the person to contact for any questions during the review process should be clearly listed.

1. Provide the following contact information for the person who is responsible for answering questions related to this Application:

Name Paul Sandoval

Title Director, PostAward, Sponsored Projects & Contracting Services

Mailing Address P.O. Box 210158, Rm 510, Tucson, AZ 85721-0158

Phone Number 520-626-6000

Email Address sponsor@email.arizona.edu

2. Legal name and address of Applicant:

Name Arizona Board of Regents, The University of Arizona

Mailing Address P.O. Box 210158, Rm 510, Tucson, AZ 85721-0158

W-9 Attached Yes

3. Name of Principal Investigator:

Name Michael Daines

Email Address mdaines@arc.arizona.edu

Date of terminal degree or completion of medical residency

01/1993

4. Title of Project

Title Impact of IVIG therapy on gut and upper airway microbiome and the associated immune response in Children's Postinfectious Autoimmune Encephalopathy

(CPAE)

5. Total Fund Request for Project Budget Period (identify dollar amount per year):

Year 1 $249,995

Year 2 $249,995

Year 3 $249,995

Total Requested $749,985

General InformationMichael Daines, ADHS BPM000334 (AZ IG) 2 of 43 mailto:mdaines@arc.arizona.edu

W-9Michael Daines, ADHS BPM000334 (AZ IG) 3 of 43

REVIEW PURPOSES

1. What field would you like your grant reviewed under? Infectious and immunological diseases and disorders

1.1. If “Other,” please specify:

Click here to enter text.

2. What is the specialized area of proposed research? Click here to enter text.

The specialized area of the proposed research is to investigate the immunomodulatory effects of IVIG on microbiome and epithelial/central immune responses. This proposal will incorporate the unique resources of the CPAE to link these changes with clinical improvement in patients treated for postinflammatory autoimmune encephalitis.

3. Does your proposed research/project include human subjects? Yes

3.1. Is your use of human subjects research exempt? No

3.1.1. If yes, please describe:

Click here to enter text.

4. Is your research an NIH defined clinical trial? No

4.1. If yes, please describe:

Click here to enter text.

5. Does the research involve Vertebrate Animals? Yes

5.1. If yes, please attach IACUC Approval Animal Welfare Assurance Number.

D16-00159

Review PurposesMichael Daines, ADHS BPM000334 (AZ IG) 4 of 43

PROJECT ABSTRACT

All Applicants are required to submit an abstract of the proposed activity suitable for publication. The abstract should be informative to other persons working in the same or related fields and, insofar as possible, understandable to a scientifically or technically literate lay reader. The abstract shall not include any proprietary or sensitive business information as it may be made available to the public.

The abstract shall be within 1,400 characters in length.

Childhood postinfectious autoimmune encephalopathies (CPAE) are diseases that impact mood, movement, and behavior in children and are being treated in the CPAE Center of Excellence at the University of Arizona. One treatment for these conditions is intravenous immunoglobulin, or IVIG, which impacts immunity and inflammation.

Understanding mechanisms behind the effects of IVIG therapy on the airway and gut microbiome and the associated immune responses may open new avenues for research into the mechanisms of immunomodulatory role of IVIG in CPAE patients specifically, and in autoimmune diseases in general. This research proposal addresses these questions in 3 aims, (1) to examine dysbiosis and dysregulation in the nasal, oral, and fecal microbiome using next generation sequencing (16S profiling and metagenomics), with particular focus on IgG-coated population of gut bacteria; (2) to examines adaptive immune responses, including pathogenic Th17 response implicated in the breakdown of the blood-brain barrier and infiltration of the relevant areas of the central nervous system by the immune cells and autoantibodies; (3) 2 pilot studies aimed to correlate IVIG-driven changes in microbiome and clinical response and a translational study to reconstitute germ-free mice with elements of the microbiome from children impacted by CPAE before and after they have been treated with IVIG.

Project AbstractMichael Daines, ADHS BPM000334 (AZ IG) 5 of 43

DETAILED PROJECT DESCRIPTION

Project Activities We have established a childhood post-infectious autoimmune encephalopathies (CPAE) Center of Excellence at the University of Arizona/Banner University Medical Group to manage PANS (pediatric acute onset neuropsychiatric syndrome) and PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections). These post-infectious inflammatory diseases that lead to alterations in mood, movement, and behavior. Most previous studies have focused on tics and OCD (obsessive-compulsive disorder) symptoms and have shown that IVIG treatment improves these symptoms. While IVIG treatment is effective, it is a complex biological substance that is expensive, available in limited supply, and there are risks and complications associated with the treatments. There are many potential mechanisms, both direct and indirect, that may lead to the potent anti-inflammatory effects of IVIG but it is unclear which mechanisms are responsible for improvement of symptoms in patients with CPAE. We will address this gap in knowledge about the mechanism of IVIG action in CPAE by studying the impact of IVIG treatment on the microbiome and epithelial immune responses. There are no published studies regarding the impact of immunomodulatory IVIG on the microbiome.

GOALS AND OBJECTIVES

Aim 1. Determine the impact of immunomodulatory IVIG on microbiome. In patients considered for IVIG treatment, we will prospectively interrogate the microbiome (nasal, oral, and fecal) using next generation sequencing (16S profiling and metagenomics), with particular focus on IgG-coated population of gut bacteria.

Goal 1.1: Determine the impact of immunomodulatory treatment with IVIG on fecal, oral, and nasal microbiome.

Objective 1.1.1 obtain microbiome samples from fecal, oral, and nasal sites before and after instituting immunomodulatory IVIG treatment. By obtaining >3 samples before and after treatment we will minimize variability and ensure the ability to assess true changes in microbiome. We anticipate enrolling an average of one subject monthly. If needed, subjects treated with immunomodulatory IVIG at other sites or for other autoimmune diseases could be included to increase the number of subjects.

Goal 1.2: It has been shown that IgG can coat fecal bacteria and this may impact their viability and function.

We will determine the impact of immunomodulatory IVIG treatment on antibody-coated fecal bacteria.

Objective 1.2.1 We will use flow cytometry and sorting to isolate IgG coated bacteria from fecal samples before and after instituting immunomodulatory IVIG treatment to identify, at a species level, which bacteria are directly impacted by IVIG treatments

Aim 2. Determine the impact of immunomodulatory IVIG on inflammation and epithelial immunity. We will study immune responses in the respiratory epithelium by non-invasive collection of the airway surface liquid for multiplexed quantification of inflammatory cytokines. Peripheral blood mononuclear cells (PBMC) will be collected and stimulated ex vivo with CD3/CD28 Dynabeads to investigate TH1, TH2, and TH17 associated cytokine production.

Goal 2.1: Cytokines have many roles in the epithelium, including proinflammatory, anti-inflammatory, and regulating cell growth. We will determine levels of epithelial cytokines and assess their modulation by IVIG treatment Objective 2.1.1 We will directly and non-invasively sample the airway surface liquid in situ in subjects before and after IVIG treatment using an absorptive but non-adsorptive sampling device. This will allow collection of undiluted fluid and the direct assessment of cytokine levels at the epithelial level.

Goal 2.2: T cells, when activated, make an array of cytokines that have powerful impacts on overall immune function and can impact epithelial immunity. T cell populations are defined by the types of cytokines they produce, including Th1 cells that produce mostly IFNg and IL12, Th2 cells that produce IL4 and IL5, and TH17 cells that produce IL17. Alterations in the overall production of these cytokines could have powerful effects on inflammation and epithelial function. To determine the impact of IVIG on more central immune responses that may also impact inflammation or epithelial responses we will also assess cytokine production by PBMCs following ex vivo stimulation Objective 2.2.1 We will obtain PBMCs from subjects before and after IVIG treatment and assess the production of inflammatory cytokines associated and determine how IVIG treatment impacts overall cytokine

Detailed Project DescriptionMichael Daines, ADHS BPM000334 (AZ IG) 6 of 43 production focusing our assessment on cytokines associated with a role impacting epithelial inflammation and function.

Aim 3. Perform two pilot studies aimed at providing preliminary data on the potentially causal relationship between IVIG, microbiota and immune and clinical response:

Pilot 3.1: Correlate changes in microbiome and inflammation/epithelial immunity after immunomodulatory IVIG with treatment response in the CPAE patients.

Goal 3.1: This aim is a pilot because it is dependent on seeing changes in microbiome or inflammatory markers following IVIG treatment in Aim 1, although we believe based on published data showing that IgG coats enteric bacteria that there will be a significant impact of IVIG on microbiome. The goal of this pilot is to correlate microbiome alteration following IVIG treatment with responses to treatment in the context of improvement in movement, mood, and behavior as assessed by a battery of standardized tests used in our clinics.

Objective 3.1.1: Because about 2/3 of patients respond to IVIG treatment and 1/3 do not, we will compare the changes in microbiome with clinical responses to treatment. We anticipate that the treatment successes will have more robust changes in microbiome than treatment failures.

Pilot 3.2: Reconstitute germ-free mice with bacteria collected from patients before and after IVIG treatment and measure changes in basal systemic and local (gut-associated lymphoid tissue) inflammatory responses.

Goal 3.2: To evaluate the causative relationship between human fecal microbiota prior and after IVIG treatment and basal mucosal inflammatory tone and epithelial barrier function in gnotobiotic mice.

Objective 3.2.1 Using established models of microbiome reconstitution in gnotobiotic mice investigate the effects of pre-IVIG and post-IVIG donor bacteria on murine barrier function and inflammation.

Background Information and Literature review

Postinfectious autoimmune reactions are well known and may represent an immune response to a pathogen’s molecular mimicry leading to a cross-reactive immune response. Anti-inflammatory treatments have been shown to be effective in mitigating these diseases. The inflammation of PANS and PANDAS has been shown to be modulatable by anti-inflammatory treatment leading to reduction in symptoms. Perlmutter et al.1 demonstrated in 1999 that IVIG and plasmapheresis both significantly reduced the symptoms of infection triggered OCD. This exceptional study was limited by focusing on tics and OCD, while other domains of cognitive function can also be impaired. In addition, while improvement in OCD scores was 45% in IVIG treated subjects and 58 percent in plasmapheresis only about 2/3 of subjects had a clinical response.

IVIG is a biologically complex substance. Each lot is made from antibodies purified from the donated plasma from thousands of humans. A liter of plasma yields 4-5 grams of IVIG, and large amounts of IVIG are needed for immunomodulatory effects, about 2 grams/kg body weight. So, an average 70kg patient would need 140 grams IVIG, requiring the donation of about 35 liters of plasma. It is expensive, with the cost of IVIG product alone at 60-200 dollars/gram.

IVIG has been used in many autoimmune diseases in addition to CPAE and there has been research regarding mechanisms of action. Mechanisms identified include impacting cytokine production, the production of T regulatory cells, inducing diversity of TCR repertoires, suppressing dendritic cell signaling, and the elimination of circulating immune complexes and cellular debris.2

Dysbiosis of the gut microbiome has been associated with and may have a causal role in a number of a number of inflammatory and autoimmune diseases including inflammatory bowel disease, metabolic syndromes, HIV associated inflammatory bowel disease, psoriasis, lupus, and rheumatoid arthritis.3 IgG is often viewed as being primarily a component of circulating plasma and not secreted. However, IgG-coated bacteria have been described in the nasopharynx of children4, middle ear effusions5, and the gut of healthy and IBD patients.6 Studies reported the ability of natural IgG to bind both gram-negative and gram-positive bacteria indirectly after the bacteria are opsonized with lectins of the innate immune system7, 8, and that gut microbiota-induced IgG

SCARED – Screening for Child Anxiety RElated Disorders Vanderbilt – ADHD CALS-Children’s Affective Lability Scale NIAS-Nine Item Avoidant/Restrictive Food Intake Disorder Screen ACE – Adverse Childhood Experiences OCI-Obsessive-Compulsive Inventory Challenging Behaviors Assessment PANESS-Physical And Neurological Examination for Soft Signs YGTSS-Yale Global Tic Severity Scale CYBOCS-Children's Yale-Brown Obsessive Compulsive Scale

Table 1. Mood, movement, and behavior testing at the CPAE Center of Excellence

Detailed Project DescriptionMichael Daines, ADHS BPM000334 (AZ IG) 7 of 43 can control systemic infection by symbiotic and pathogenic bacteria.9 We hypothesize that therapeutic doses of immunomodulatory IVIG can impact all of these responses by modulating immune responses from GALT and dendritic cells and by directly binding to gut bacteria.

PRELIMINARY WORK, EXPERIMENTAL METHODOLOGY, AND DATA ANALYSIS

Aim 1. Determine the impact of immunomodulatory IVIG on microbiome.

Preliminary work

In our preliminary observations, gut microbiome of CPAE-affected children varies from healthy controls. To exemplify this, Figure 1 shows microbial taxonomic differences in fecal samples of identical twins discordant for CPAE, i.e. patients sharing not only genetic identity, but also the same environment and nutritional status.

We hypothesize that IVIG therapy will result in higher level of IgG-coated bacteria and propose to identify the changes in the microbial community structure in the gut and at accessible mucosal surfaces in children and that luminal IgG increases in the gut and coats a specific population if microbes to regulate mucosal and systemic immune responses.

Methodology

Sample collection and storage Nasal and throat samples will be collected with OMNIgene ORAL swabs in the clinic, de-identified, coded, and transferred immediately to the research lab for storage and DNA isolation. Stools will be collected by the patients’ parents at home using the provided kit. Parents will place the collection container in the plastic holder, before placing it underneath the toilet seat. Once study participant has finished, parents will use precautionary measures, including proper handwashing techniques and gloves, to remove the collection container from the toilet. Before the container is firmly sealed, parent will sample a small amount of stool with a provided scoop and place it into a dedicated tube (OMNIgene GUT sample collection tube provided with the kit) with a DNA preservative solution. Both samples will be paced in a provided Styrofoam box and returned to study staff for specimen processing. The samples will de-identified, coded, and transported to and stored in a secure lab at the College of Medicine. Stabilized DNA from OMNIgene GUT tubes will be used for DNA isolation, while the bulk stool sample will be stored at -80oC until used for flow sorting and colonization of germ-free mice (Aim 3, Pilot 3.2).

Because of variability in the fecal microbiome and because many of these children are on antibiotics, we will collect 3 stool samples prior to IVIG and 3 stool samples after IVIG

Flow sorting of IgG-coated bacteria From the bulk samples, aliquots will be used for flow sorting of IgG-coated bacteria. Briefly, after clearing the fecal debris by centrifugation, bacteria will be stained with FITC-conjugated anti-human IgG Fc (Abcam) and BacLight™ Red Bacterial Stain (Thermo Fisher) and evaluated via flow cytometry. IgG-coated bacteria will be flow sorted using BD FACSARIA III, and DNA extracted for metagenomic analysis. Kiela lab has optimized the protocol for isolation of IgA-coated bacteria in mouse samples (Figure 2) and the same principle will be applied here.

Visual Motor Integration Beery VMI-6: VMI Perception Beery VMI-6: VP

Graphomotor Berry VMI-6: MC Executive Function D-KEFS Color-Word Interference Test

Verbal Memory WRAML-2: Verbal Learn.

VL Delay Recall VL Recognition

Nonverbal IQ WPPSI-IV Matrix R.

WASI-II Matrix R.

Processing Speed (motor heavy) WNV Coding WISC-V Coding fine-motor Purdue Pegboard

Verbal IQ WPPSI-IV Vocab WASI-II Vocab

Auditory Working Memory DAS-II Digit Fwd DAS-II Digit Bkwd WISC-V Digit Span

Processing Speed (Less motor) WPPSI-IV Bug Search WISC-V Symbol Search

Reading WRAT-5 Word Reading Math WRAT-5 Math Computation

Symptom Validity Memory Validity Profile Table 2. Psychometric testing performed on all subjects before and after immunomodulatory IVIG

Genus

Fig 1. Taxonomic analysis at the genus level of fecal microbial composition of identical twins discordant for CPAE.

Detailed Project DescriptionMichael Daines, ADHS BPM000334 (AZ IG) 8 of 43

16S amplicon profiling Samples of oral, nasal, and fecal bacterial DNA will be used for 16S-basewd community composition analysis. The hypervariable V4 region of the 16S rRNA gene will be amplified from each sample using barcoded primers consisting of Golay barcodes, sequencing primer pads, linkers, and Illumina adapters10, and with MyFi polymerase Master Mix (Bioline). Amplicons will be quantified using Quant-It PicoGreen dsDNA Assay kit (Invitrogen), according to the manufacturer’s protocol. Equal amount of amplified DNA (240ng) from each sample will be pooled and cleaned using UltraClean PCR Clean-Up Kit (MoBio). Pooled amplicons are then diluted, denatured and sequenced on MiSeq platform (Illumina) using custom primers (Caporaso et al., 2012). Due to the limited sequence diversity among 16S rRNA amplicons, 5% of the PhiX control library (Illumina) made from phiX174, is added to the run. The pooled 16S rRNA library will be subjected to the paired-end sequencing using 2 x 150bp MiSeq Reagent Kit V2 (Illumina). Sequencing of all samples was performed at our laboratory on Illumina MiSeq and fastq files were generated.

De-multiplexing was done using idemp script. Filtering, dereplication, sample inference, chimera identification, and merging of Paired-end reads was done with a reference free Divisive Amplicon Denoising Algorithm 2 (Dada2) R package (Callahan et al., 2016). The ASVs taxonomy was assigned using RDP classifier against SILVA database release 132. The vegan package, a tool for diversity analysis, ordination methods, for the analysis of dissimilarities and statistical analysis will be used. Obtained results will be visualized with ggplot2 package in the R environment.

Data Analysis

Metagenomics High- throughput sequencing shotgun metagenomic technique allows to analyze whole microbial genomes of microbial communities in a random, untargeted manner. This method allows to analyze in depth microbial composition as well as analyze its metabolic capability, presence of specific metabolic pathways or antibiotic resistance genes. This method will be applied to analyze the microbial targets coated with IgG in the fecal samples using DAN from flow-sorted IgG+ bacteria. For library preparation, QIAseq FX DNA library Kit (Qiagen) will be used according to the manufacturer’s protocol. Briefly, extracted genomic DNA extracted is enzymatically sheared into smaller 250-280bp fragments, followed by platform specific adapter ligation. After PCR-based amplification, dual-barcoded library is purified with AMPure XP magnetic beads and unused primers are removed. The DNA concentration is quantified using qPCR-based method and the quality assess using a capillary electrophoresis device such as Agilent BioAnalyzer. Normalized and pooled library is sequenced on Illumina NextSeq 550 platform using either Mid Output or High Output sequencing kit depend on the number of samples and the required coverage. FastQC tool is used to provide quality check of the demultiplexed fastq files and raw sequence data. FastQC is a modular tool providing QC information about total count of reads, filtering, sequence length distribution, percent of GC content, information about duplicate sequences, and overrepresented sequences and Kmers. An assembly-free metagenome profiling will be used, and the direct mapping approach will be implemented to estimate the functional gene composition of each sample. Reads are mapped against functional reference sequence databases using Bowtie2 (Langmead and Salzberg, 2012) in MethaPhlAn, a metagenomic phylogenetic analysis tool for microbial community profiling.

Diversity parameters will be estimated from functional gene matrices that were rarefied to an equal number per sample. Statistical comparison of functional gene richness among samples will be performed by Mann– Whitney U test. Statistical comparison of functional gene composition among samples will be performed by Permutational multivariate analysis of variance (PERMANOVA) with popular R packages, such as DESeq2 or vegan. Data will be visualized using R packages, e.g. ggplot2.

Fig. 2. Flow cytometry analysis of murine IgA-coated bacteria. Gated population was used for sorting and DNA isolation.

Detailed Project DescriptionMichael Daines, ADHS BPM000334 (AZ IG) 9 of 43

Aim 2. Determine the impact of immunomodulatory IVIG on inflammation and epithelial immunity.

Methodology and Data Analysis

Cytokine analysis Samples of the upper respiratory tract fluid, and cell culture supernatant from patients before and after IVIG therapy will be analyzed using multiplexed xMap assay and MagPix instrument (Luminex; available in the Kiela lab). We will analyze 41 chemokines/cytokines in a panel allowing for sensitive discrimination between Th1, Th2, and Th17 responses with a small volume (20 l) of samples (MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel; Millipore). Quantitation will be performed with the MILLIPLEX® Analyst 5.1 software with curve fitting algorithms that optimize mathematical methods to minimize fitting errors.

Final date will be analyzed with paired T-test or repeated measures ANOVA, as indicated, and visualized using Prism 8 software (Graphpad).

Aim 3. Perform two pilot studies aimed at providing preliminary data on the potentially causal relationship between IVIG, microbiota and immune and clinical response:

Methodology and Data Analysis

Pilot 3.1: Correlate changes in microbiome and inflammation/epithelial immunity after immunomodulatory IVIG with treatment response in the CPAE patients.

As shown in Table 1 and Table 2, children seen at the CPAE have extensive testing of cognitive function, mood, movement, and behavior.11-15 These validated assessment tools allow us to define the functional problems in these children and objectively assess treatment responses. We will classify children as responders if they have a substantial (>1 st dev on neuropsychiatric testing or a clinically relevant response to the other tests) improvement after IVIG treatment. We will compare the inflammatory responses and microbiome changes associated with IVIG treatment in IVIG responders and non-responders. An ANOVA or t test with correction for multiple observations will be performed. Additionally, we will use the improvement in testing results as continuous variables to look for association with microbiome and inflammatory changes

Pilot 3.2: Reconstitute germ-free mice with bacteria from humans collected before and after IVIG treatment and measure changes in basal systemic and local (gut-associated lymphoid tissue) inflammatory responses.

Within its limitations, germ-free mice offer unique model to transcend clinical correlations and test for causal relationships between altered microbiome and immune and other physiological functions.16 Kiela lab has successfully created gnotobiotic mice harboring human microbiome from critically ill pediatric patients with varying degree of gut barrier function, and demonstrated not only relatively faithful colonization, but also partial phenotype transfer with varying degree of colitis (Typpo et al., manuscript in preparation). We will apply the same technique, and with the help with Dr. Karuna Patil (University Animal Care), we will associate germ-free C57BL/6 mice with fecal slurries prepared from CPAE patients prior to and after IVIG therapy. We postulate that these defined and likely distinct communities will exert different effects on the basal immune/inflammatory tone using murine gut as a model target. Briefly, GF mice will be colonized with fecal slurries prepared from , extraction and 16S profiling (input) and ca. 250mL of the slurries will be gavaged orally, and similar amount will be used to “paint” the abdominal surface. Mice will then be placed individually in Techniplast Iso-Positive ventilated cages (see descriptions of animal facilities) and maintained for 3 weeks until euthanized. Colonic content will be collected for 16S amplicon profiling (see methods above), and sections of the ileum, proximal, and distal colon will be removed for H&E staining and histological scoring, RNA extraction and qPCR analysis of cytokine and chemokine profiles, and for permeability analysis in Ussing chambers (available and used routinely in the Kiela lab). We will focus qPCR analysis on key mucosal inflammatory mediators, such as IL1b, IL6, TNF, IFNg, IL17a, IL22, IL4, KC, Ptgs2, MIP-2, MMP8 (as neutrophil marker). Ussing chamber experiments will determine segment-specific transepithelial resistance (TEER) and transmucosal flux of FITC-labeled dextran (FD4, Sigma-Aldrich).

Detailed Project DescriptionMichael Daines, ADHS BPM000334 (AZ IG) 10 of 43

TIMELINE

This 3-year project is outlined in Table 3. We anticipate enrolling subjects throughout the entire duration of the project, collecting and analyzing samples for microbiome and inflammation contemporaneously.

Reconstitution of gnotobiotic mice in pilot 3.1 will be done in year 3. Viable bacteria can be stored and then once sufficient samples are collected in year 1 and 2 used to reconstitute these mice.

We will assess enrollment every 6 months. If we do not enroll at the anticipated rate we will expand to include enrolling subjects from other institutions treating CPAE (we are part of a network of 22 institutions, linked by the NIH/NIMH, that would be potential partners for increasing subjects). We can also enroll subjects getting IVIG for other inflammatory diseases because the immunomodulatory effects for these treatments would be identical.

EVALUATION

Key personnel already have ongoing group meetings as part of the research projects for the CPAE. We will continue these meetings and review enrollment, sample collection, and data quality for all objectives in Aim 1 and Aim 2.

We will have quarterly interim reviews where we will assess the need to expand recruitment based on enrollment and assess the correlation between clinical responses to treatment and the inflammatory markers and microbiome changes (Pilot Aim 3.1).

We will review the gnotobiotic mouse experiments in Pilot Aim

3.2 during the third year of the project. These experiments will be simultaneously rather than as the samples are collected to minimize variability inherent in mouse experiments.

Publication will allow outside independent reviews of data, we anticipate publishing at least one manuscript from each Aim.

IRB will assist in monitoring patient safety, although this is a non-interventional study so there should be no patient safety impact.

Every 6 months we will present our data to group laboratory meetings to get intramural review and suggestions for optimizing the project. Every 6 months we will meet with a biostatistician to review data analysis strategy and results.

Table 3. Timeline of experiments Year 1 Enroll subjects Collect samples for microbiome analysis Collect samples for inflammatory analysis Analyze samples Statistical comparisons, pre-and post-IVIG Year 2 Enroll subjects Collect samples for microbiome analysis Collect samples for inflammatory analysis Analyze samples Statistical comparisons, pre-and post-IVIG Expand to include other sites or subjects getting IVIG for non-CPAE reasons if slow recruitment Year 3 Enroll subjects Collect samples for microbiome analysis Collect samples for inflammatory analysis Analyze samples Statistical comparisons, pre-and post-IVIG Reconstitute mice with pre-and post-IVIG samples and assess inflammatory alterations Statistical correlations between clinical responses to IVIG and inflammatory or microbiome responses to IVIG

Detailed Project DescriptionMichael Daines, ADHS BPM000334 (AZ IG) 11 of 43

Project resources: Overall: Multispecialty-multidisciplinary research and outreach present unique challenges that can only be addressed by comprehensive clinical and research resources. At the University of Arizona we have established a CPAE that has unique resources to address these challenges using support from many private and public entities including Banner University Medical Center-Tucson, the College of Medicine, the State of Arizona, the Steele Center, and the PACE foundation. Our clinical and clinical-translational research staff include Michael Daines, MD (Immunology), Sydney Rice, (Developmental Pediatrics), Sejal Jain, MD (pediatric neurology), Peter Klinger, MD (child psychiatry), Andrew Gardner, PHD, BCBA (behavior), Dan Combs, MD (sleep), Fayez Ghishan, MD (Pediatric Gastroenterology and a consultant for animal models and translational work in inflammatory research, letter of support attached), Jessica West, MS, RN and technicians for the proposed testing. Our research team includes Pawel Kiela, DVM, PHD, Jennifer Andrews, PHD, Maureen Galindo, MS, RN, and Matthew Murray, BS. Through the Steele center we have access to database, computational, and statistical support to ensure success of this proposal.

Laboratory: Dr. Kiela’s laboratory (ca. 3,000 sq.) is located within the Steele Children's Research Center of the University of Arizona College of Medicine. It is very well equipped to carry the proposed studies including both basic research equipment (e.g. pipettes, plastic and glass lab-ware, benchtop centrifuges, electrophoresis apparatus, gel documentation system, cell culture facilities, incubators, microscopes, etc.), as well as more sophisticated, application-specific instrumentation (see Major Equipment below).

As a member of the NCI-accredited comprehensive University of Arizona Cancer Center Arizona, Dr. Kiela has also access the resources at The Genomics Shared Resource (GSR), which among other paltforms and services, offers two Ion Torrent PGM sequencing platforms capable of performing the 16S amplicon metagenomics sequencing as described in the preliminary data and Research Strategy.

Animal Facility/Gnotobiotic Facility: AALAC approved University Animal Care (UAC) is under the leadership of Dr. David G. Besselsen, DVM., Ph.D, a long-term collaborator of Dr. Kiela’s and an experienced veterinary pathologist. UAC is an animal care research center with facilities located at the University of Arizona Health Science Center (AHSC), main campus Central Animal Facility (CAF), and an ultraclean barrier facility located in the adjacent Bio5 building. Its primary mission is to assure that healthy animals are available for use by faculty members and students who have received approval to perform research, testing, or educational studies on animal subjects. UAC ensures humane and appropriate animal care and use by providing for the animal's daily care needs, providing necessary veterinary care, monitoring use of animals by principal investigators and their technical personnel, and by ensuring that the highest standards are met.

Bio-5 animal center is the University’s strictest mouse barrier vivarium which includes a dedicated germ-free/gnotobiotic (UAGF) facility, which occupies two adjacent rooms and currently houses 14 flexible-film isolators and 80 Techniplast Iso-Positive ventilated cages (o maintain and/or utilize for experimentation germ-free and gnotobiotic mice. The facility is led by Dr. Karuna Patil, DVM, MS, DACLAM, Associate Veterinary Specialist with considerable experience with gnotobiotics. Dr. Patil and her trained staff is in charge of rederivation of mouse strains to germ-free status, their colonization with unique microbiota of interest, and performing experimental manipulations common in GF/GN mouse experiments (gavage, injections, sample collection, body weight, body condition scores, etc.).

Isolator sterility is monitored on a weekly basis by cultivation dependent methods for bacterial and mycotic contamination. This includes culture of drinking water and fresh fecal samples from animals housed in each isolator in three different mediums (Nutrient, SabDex, and BHI broth) in both aerobic and anaerobic environments. Additionally, at monthly intervals, cultivation independent (16S PCR) is performed on each isolator. Strict standard operating procedures are followed for introduction of materials into isolators including isolator to isolator animal transfer. These procedures, which utilize Clidox-S (1:3:1 concentration), make the risk of contamination from viral and parasitic agents extremely low and therefore no virology or parasitology has been performed on our germ-free populations to date.

University of Arizona Gnotobiotic Mouse Facility maintains a stable colony of WT C57BL/6, Swiss -Webster and Balb/c mice, and provides them to the investigators as needed. Serviced and procedures are also in place for mouse strain rederivation into the germ-free environment through the cesarean technique.

Techniplast Iso-Positive ventilated (ISO) cages. ISO cages feature twin clamps and double gasket self-closing nozzles; they are airtight, hermetic cages, equipped with individual HEPA filters and their own filtered positive airflow. These cages require less space and reduce animal housing costs, while allowing for the maintenance of the GF status for at least 12 weeks and provide an environment to maintain stable microbiome after fecal microbiome transplant (Paik et al. Gut Microbes. 2015 Jul 4;6(4):255-65). The Air Handling Unit has a Pressure driven microprocessor based ventilation control that guarantees constant high positive pressure to provide a safe intra-cage environment.

Detailed Project DescriptionMichael Daines, ADHS BPM000334 (AZ IG) 12 of 43

6. Major Equipment: Dr. Kiela’s lab is well-equipped to conduct the proposed studies. Major equipment includes luminometer, thermal cyclers, plate spectrophotometer, plate fluorometer, 17-parameter flow cytometer (BD LSRFortessa), MagPix, fluorescent and laser confocal microscopes, confocal laser cytometer, etc.), Affymetrix Gene Chip system with data analysis workstation, MiSeq sequencer (Illumina) with data analysis workstation [Apple MacPro, 2.7GHz 12-Core Intel Xeon E5 (24 logical cores), 128GB RAM] with backup data storage. The lab is equipped with a 4-color laser confocal microscope (Olympus FV10i) with computer workstation and deconvolution software (Huygens; Scientific Volume Imaging b.v., Netherlands), and an epifluorescent automated microscope with incubated stage (EVOS FL Auto). A cold room, two tissue culture rooms with four cell culture hoods and four water jacketed CO2 incubators, a common equipment room with three -80oC freezers (two more available in adjacent lab) and liquid N2 storage, two ultracentrifuges, shaking and stationary bacterial incubators, designated work areas (dark room with X-ray film developer, and a dedicated room for radioisotope use). The lab is also equipped with an BactronEZ anaerobic chamber (Sheldon Manufacturing, Inc.) for bacterial culture, with the capacity of 12.5 Cu.Ft., capable of holding 300 petri dishes. As a member of the NCI-accredited comprehensive University of Arizona Cancer Center Arizona, Dr. Kiela has also access the resources at The Genomics Shared Resource (GSR), which among other platforms and services, offers two Ion Torrent PGM sequencing platforms capable of performing the 16S amplicon metagenomics sequencing.

Clinical: Banner University Medical Center provides dedicated facilities for the clinical component of the Children’s Postinfectious Autoimmune Encephalopathy (CPAE) Center of Excellence.

Computer: The laboratories are equipped with a total of 15 computers ranging from Pentium 4 2.8 GHz to Pentium i7 3.8 GHz desktop computers. Laser and ink-jet printers are available in the investigators laboratory. PI's office is equipped with Apple iMac 3.2GHz Intel i5 desktop and 2016 MacBook Pro. All computers are linked to the Internet through a high-speed LAN (100 Mbs or 1 Gbs). Office productivity software and more specialized data analysis and statistical software are available to all investigators. Dr.

Kiela has licenses for GeneSpring microarray analysis software (Agilent) and IPA Pathway Analysis software (Qiagen). IT infrastructure and support services are provided by UA College of Medicine Information Technology Services (COM ITS). COM ITS adheres to the information security policies, standards, procedures and guidelines published by the University of Arizona. Backup service through secure network attached storage is in place. Researchers in the PI’s group share files via Dropbox Pro and Box cloud storage accounts.

5. Office: Dr. Kiela has dedicated office space of 120 sq. ft. available located near his research laboratory.

Dr. Rice has dedicated office space located at an offsite University of Arizona owned property. Dr. Daines has dedicated office space in the Steele Children’s Research tower.

Other:

The University of Arizona: Founded in 1885, the University of Arizona is the leading public research university in the American Southwest and an ideal transdisciplinary research community for the proposed work. A land- grant, state university governed by the Arizona Board of Regents, the University produces more than $625 million in annual research and is the only Arizona university belonging to the prestigious 62-member Association of American Universities. It ranks 18th among public universities according to the National Science Foundation. Enrolling more than 42,000 students representing every state in America and over 100 other countries in over 350 degree fields, this is a diverse community of people who thrive on innovation and collaboration, an amazing environment for interacting with students, faculty and researchers from many diverse disciplines.

In summary, the College of Medicine offers a strong and supportive environment for research. The PIs have full access to all shared instrumentation within the Steele Children’s Research Center at the Arizona Health Sciences Center. Several very well equipped and staffed University-wide core facilities are available on a fee-per-service basis. Many of them are affiliated with the Arizona Cancer Canter (adjacent to AHSC), of which the PI is a full member. Links to all shared facilities available to the UA researchers are listed at http://research.arizona.edu/core- facilities. The University of Arizona Microscopy Alliance is a collaborative effort to bring information about shared microscopy facilities to the University of Arizona and the community.

All available resources are listed at http://microscopy.arizona.edu/.

Detailed Project DescriptionMichael Daines, ADHS BPM000334 (AZ IG) 13 of 43

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2. Martinez T, Garcia-Robledo JE, Plata I, Urbano MA, Posso-Osorio I, Rios-Serna LJ et al. Mechanisms of action and historical facts on the use of intravenous immunoglobulins in systemic lupus erythematosus. Autoimmun Rev 2019; 18(3): 279-286.

3. Fung TC, Olson CA, Hsiao EY. Interactions between the microbiota, immune and nervous systems in health and disease. Nat Neurosci 2017; 20(2): 145-155.

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5. Stenfors LE, Raisanen S. Secretory IgA- and IgG-coated bacteria in chronically discharging ears. J Laryngol Otol 1991; 105(7): 515-517.

6. Harmsen HJ, Pouwels SD, Funke A, Bos NA, Dijkstra G. Crohn's disease patients have more IgG-binding fecal bacteria than controls. Clin Vaccine Immunol 2012; 19(4): 515-521.

7. Puga I, Cerutti A. Protection by natural IgG: a sweet partnership with soluble lectins does the trick!

EMBO J 2013; 32(22): 2897-2899.

8. Panda S, Zhang J, Tan NS, Ho B, Ding JL. Natural IgG antibodies provide innate protection against ficolin-opsonized bacteria. EMBO J 2013; 32(22): 2905-2919.

9. Zeng MY, Cisalpino D, Varadarajan S, Hellman J, Warren HS, Cascalho M et al. Gut Microbiota- Induced Immunoglobulin G Controls Systemic Infection by Symbiotic Bacteria and Pathogens.

Immunity 2016; 44(3): 647-658.

10. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 2012; 6(8): 1621- 1624.

11. Birmaher B, Brent DA, Chiappetta L, Bridge J, Monga S, Baugher M. Psychometric properties of the Screen for Child Anxiety Related Emotional Disorders (SCARED): a replication study. J Am Acad Child Adolesc Psychiatry 1999; 38(10): 1230-1236.

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21(4): 773-800.

13. Lewin AB, Piacentini J, De Nadai AS, Jones AM, Peris TS, Geffken GR et al. Defining clinical severity in pediatric obsessive-compulsive disorder. Psychol Assess 2014; 26(2): 679-684.

14. Shaffer D, Gould MS, Brasic J, Ambrosini P, Fisher P, Bird H et al. A children's global assessment scale (CGAS). Arch Gen Psychiatry 1983; 40(11): 1228-1231.

15. Hays JR, Reas DL, Shaw JB. Concurrent validity of the Wechsler abbreviated scale of intelligence and the Kaufman brief intelligence test among psychiatric inpatients. Psychol Rep 2002; 90(2): 355-359.

16. Nguyen TL, Vieira-Silva S, Liston A, Raes J. How informative is the mouse for human gut microbiota research? Dis Model Mech 2015; 8(1): 1-16.

ReferencesMichael Daines, ADHS BPM000334 (AZ IG) 14 of 43

OMB No. 0925-0001 and 0925-0002 (Rev. 09/17 Approved Through 03/31/2020)

BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

NAME: DAINES, MICHAEL O

eRA COMMONS USER NAME (agency login): MDAINE POSITION TITLE: Associate Professor of Pediatrics, Division Chief Pediatric Allergy, Immunology, and Rheumatology. Co-director Center for Pediatric Autoimmune Encephalopathy EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)

INSTITUTION AND LOCATION DEGREE

(if applicable)

Completion Date

MM/YYYY

FIELD OF STUDY

Texas A&M University BS 01/1989 St. Louis University School of Medicine MD 01/1993 Cardinal Glennon Children's Hospital, St. Louis, MO

Resident 06/1996 Pediatrics Residency

Duke University Medical Center, Durham, NC

Fellow 06/1998 Pediatric Allergy Immunology Fellowship

A. Personal Statement As a pediatric allergist-immunologist, I have a strong interest in improving health outcomes in children with inflammatory diseases such as postinfectious autoimmune encephalopathy and understanding how inflammatory dysregulation can lead to these responses. Since completing my fellowship in allergy/immunology, I have focused my academic efforts on basic, translational, and clinical research focusing on inflammation with a particular emphasis on host-allergen and host-pathogen interaction. My research began at Cincinnati Children’s hospital, where I was given the research support and mentorship to flourish with a successful K08 application studying IL13Ra2 and its related signaling pathways. This initial research allowed me to develop expertise in animal and cell-based techniques and expand my research into more translational directions. I continued to study allergen proteases and their interactions with host protease defenses as PI on an R01 grant since coming to the University of Arizona College of Medicine. I have strong research ties here, with multi-year collaborations with many investigators. My strong background in basic and translational inflammatory research and my skill at working with clinical trials make me uniquely suited to be one of the leaders of the CPAE research into causes of PANS (Pediatric Acute-onset Neuropsychiatric Syndrome) and PANDAS (Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections) and to determine the optimal way to assess treatment responses.

a. Daines MO, Hershey GK. A novel mechanism by which interferon-gamma can regulate interleukin (IL)-13 responses. Evidence for intracellular stores of IL-13 receptor alpha -2 and their rapid mobilization by interferon-gamma. J Biol Chem. 2002 Mar 22;277(12):10387-93.

PubMed PMID: 11786536.

b. Daines MO, Chen W, Tabata Y, Walker BA, Gibson AM, Masino JA, Warrier MR, Daines CL, Wenzel SE, Hershey GK. Allergen-dependent solubilization of IL-13 receptor alpha2 reveals a novel mechanism to regulate allergy. J Allergy Clin Immunol. 2007 Feb;119(2):375-83. PubMed

PMID: 17140645.

c. Sivaprasad U, Askew DJ, Ericksen MB, Gibson AM, Stier MT, Brandt EB, Bass SA, Daines MO, Chakir J, Stringer KF, Wert SE, Whitsett JA, Le Cras TD, Wills-Karp M, Silverman GA, Khurana Hershey GK. A nonredundant role for mouse Serpinb3a in the induction of mucus

Biographical SketchesMichael Daines, ADHS BPM000334 (AZ IG) 15 of 43 http://www.ncbi.nlm.nih.gov/pubmed/11786536/ http://www.ncbi.nlm.nih.gov/pubmed/17140645/ production in asthma. J Allergy Clin Immunol. 2011 Jan;127(1):254-61, 261.e1-6. PubMed PMID: 21126757; PubMed Central PMCID: PMC3058372.

d. Boitano S, Flynn AN, Sherwood CL, Schulz SM, Hoffman J, Gruzinova I, Daines MO. Alternaria alternata serine proteases induce lung inflammation and airway epithelial cell activation via PAR2. Am J Physiol Lung Cell Mol Physiol. 2011 Apr;300(4):L605-14. PubMed PMID:

21296894; PubMed Central PMCID: PMC3075100.

B. Positions and Honors Positions and Employment 2002 - 2004 Research Instructor, Cincinnati Children's Hospital Medical Center 2004 - 2007 Assistant Professor of Pediatrics, Cincinnati Children's Hospital Medical Center 2007 - 2011 Assistant Professor of Pediatrics, University of Arizona College of Medicine 2011- 2016- 2016-

Associate Professor of Pediatrics, University of Arizona College of Medicine Division Chief, Pediatric Allergy, Immunology, and Rheumatology Co-director, Center for Pediatric Autoimmune Encephalopathy

Other Experience and Professional Memberships 1999- Member, American Academy of Asthma, Allergies, and Immunology 2003- Member, American Association of Immunologists 2005- Member, American Academy of Allergy, Asthma and Immunology, Genetics, Molecular Biology & Epidemiology Committee 2007- Member, Venom Immunochemistry, Pharmacology and Emergency Response

(VIPER) Institute ARC 2015- Standing…