A replicon RNA vaccine can induce durable protective immunity from SARS-CoV-2 in nonhuman primates after neutralizing antibodies have waned.

The global SARS-CoV-2 pandemic prompted rapid development of COVID-19 vaccines. Although several vaccines have received emergency approval through various public health agencies, the SARS-CoV-2 pandemic continues. Emergent variants of concern, waning immunity in the vaccinated, evidence that vaccines may not prevent transmission and inequity in vaccine distribution have driven continued development of vaccines against SARS-CoV-2 to address these public health needs. In this report, we evaluated a novel self-amplifying replicon RNA vaccine against SARS-CoV-2 in a pigtail macaque model of COVID-19 disease. We found that this vaccine elicited strong binding and neutralizing antibody responses against homologous virus. We also observed broad binding antibody against heterologous contemporary and ancestral strains, but neutralizing antibody responses were primarily targeted to the vaccine-homologous strain. While binding antibody responses were sustained, neutralizing antibody waned to undetectable levels in some animals after six months but were rapidly recalled and conferred protection from disease when the animals were challenged 7 months after vaccination as evident by reduced viral replication and pathology in the lower respiratory tract, reduced viral shedding in the nasal cavity and lower concentrations of pro-inflammatory cytokines in the lung. Cumulatively, our data demonstrate in pigtail macaques that a self-amplifying replicon RNA vaccine can elicit durable and protective immunity to SARS-CoV-2 infection. Furthermore, these data provide evidence that this vaccine can provide durable protective efficacy and reduce viral shedding even after neutralizing antibody responses have waned to undetectable levels.

A decay-modeled compressed sensing reconstruction approach for non-Cartesian hyperpolarized 129Xe MRI.

PURPOSE: Hyperpolarized 129Xe MRI benefits from non-Cartesian acquisitions that sample k-space efficiently and rapidly. However, their reconstructions are complex and burdened by decay processes unique to hyperpolarized gas. Currently used gridded reconstructions are prone to artifacts caused by magnetization decay and are ill-suited for undersampling. We present a compressed sensing (CS) reconstruction approach that incorporates magnetization decay in the forward model, thereby producing images with increased sharpness and contrast, even in undersampled data. METHODS: Radio-frequency, T1, and T 2 * $$ {\mathrm{T}}_2^{\ast } $$ decay processes were incorporated into the forward model and solved using iterative methods including CS. The decay-modeled reconstruction was validated in simulations and then tested in 2D/3D-spiral ventilation and 3D-radial gas-exchange MRI. Quantitative metrics including apparent-SNR and sharpness were compared between gridded, CS, and twofold undersampled CS reconstructions. Observations were validated in gas-exchange data collected from 15 healthy and 25 post-hematopoietic-stem-cell-transplant participants. RESULTS: CS reconstructions in simulations yielded images with threefold increases in accuracy. CS increased sharpness and contrast for ventilation in vivo imaging and showed greater accuracy for undersampled acquisitions. CS improved gas-exchange imaging, particularly in the dissolved-phase where apparent-SNR improved, and structure was made discernable. Finally, CS showed repeatability in important global gas-exchange metrics including median dissolved-gas signal ratio and median angle between real/imaginary components. CONCLUSION: A non-Cartesian CS reconstruction approach that incorporates hyperpolarized 129Xe decay processes is presented. This approach enables improved image sharpness, contrast, and overall image quality in addition to up-to threefold undersampling. This contribution benefits all hyperpolarized gas MRI through improved accuracy and decreased scan durations.

Highly accelerated dynamic acquisition of 3D grid-tagged hyperpolarized-gas lung images using compressed sensing.

PURPOSE: To develop and test compressed sensing-based multiframe 3D MRI of grid-tagged hyperpolarized gas in the lung. THEORY AND METHODS: Applying grid-tagging RF pulses to inhaled hyperpolarized gas results in images in which signal intensity is predictably and sparsely distributed. In the present work, this phenomenon was used to produce a sampling pattern in which k-space is undersampled by a factor of approximately seven, yet regions of high k-space energy remain densely sampled. Three healthy subjects received multiframe 3D 3 He tagging MRI using this undersampling method. Images were collected during a single exhalation at eight timepoints spanning the breathing cycle from end-of-inhalation to end-of-exhalation. Grid-tagged images were used to generate 3D displacement maps of the lung during exhalation, and time-resolved maps of principal strains and fractional volume change were generated from these displacement maps using finite-element analysis. RESULTS: Tags remained clearly resolvable for 4-6 timepoints (5-8 s) in each subject. Displacement maps revealed noteworthy temporal and spatial nonlinearities in lung motion during exhalation. Compressive normal strains occurred along all three principal directions but were primarily oriented in the head-foot direction. Fractional volume changes displayed clear bilateral symmetry, but with the lower lobes displaying slightly higher change than the upper lobes in 2 of the 3 subjects. CONCLUSION: We developed a compressed sensing-based method for multiframe 3D MRI of grid-tagged hyperpolarized gas in the lung during exhalation. This method successfully overcomes previous challenges for 3D dynamic grid-tagging, allowing time-resolved biomechanical readouts of lung function to be generated.

Sonoselective transfection of cerebral vasculature without blood-brain barrier disruption.

Treatment of many pathologies of the brain could be improved markedly by the development of noninvasive therapeutic approaches that elicit robust, endothelial cell-selective gene expression in specific brain regions that are targeted under MR image guidance. While focused ultrasound (FUS) in conjunction with gas-filled microbubbles (MBs) has emerged as a noninvasive modality for MR image-guided gene delivery to the brain, it has been used exclusively to transiently disrupt the blood-brain barrier (BBB), which may induce a sterile inflammation response. Here, we introduce an MR image-guided FUS method that elicits endothelial-selective transfection of the cerebral vasculature (i.e., "sonoselective" transfection), without opening the BBB. We first determined that activating circulating, cationic plasmid-bearing MBs with pulsed low-pressure (0.1 MPa) 1.1-MHz FUS facilitates sonoselective gene delivery to the endothelium without MRI-detectable disruption of the BBB. The degree of endothelial selectivity varied inversely with the FUS pressure, with higher pressures (i.e., 0.3-MPa and 0.4-MPa FUS) consistently inducing BBB opening and extravascular transfection. Bulk RNA sequencing analyses revealed that the sonoselective low-pressure regimen does not up-regulate inflammatory or immune responses. Single-cell RNA sequencing indicated that the transcriptome of sonoselectively transfected brain endothelium was unaffected by the treatment. The approach developed here permits targeted gene delivery to blood vessels and could be used to promote angiogenesis, release endothelial cell-secreted factors to stimulate nerve regrowth, or recruit neural stem cells.

Profiling of the immune landscape in murine glioblastoma following blood brain/tumor barrier disruption with MR image-guided focused ultrasound.

PURPOSE: Glioblastoma (GB) poses formidable challenges to systemic immunotherapy approaches owing to the paucity of immune infiltration and presence of the blood brain/tumor barriers (BBB/BTB). We hypothesize that BBB/BTB disruption (BBB/BTB-D) with focused ultrasound (FUS) and microbubbles (MB) increases immune infiltration in GB. As a prelude to rational combination of FUS with ITx, we herein investigate the impact of localized BBB/BTB-D on innate and adaptive immune responses in an orthotopic murine GB model. METHODS: Mice with GL261 gliomas received i.v. MB and underwent FUS BBB/BTB-D (1.1 MHz, 0.5 Hz pulse repetition frequency, 10 ms bursts, 0.4-0.6 MPa). Brains, meninges, and peripheral lymphoid organs were excised and examined by flow cytometry 1-2 weeks following FUS. RESULTS: The number of dendritic cells (DC) was significantly elevated in GL261 tumors and draining cervical LN in response to sonication. CD86 + DC frequency was also upregulated with 0.6 MPa FUS, suggesting increased maturity. While FUS did not significantly alter CD8 + T cell frequency across evaluated organs, these cells upregulated checkpoint molecules at 1 week post-FUS, suggesting increased activation. By 2 weeks post-FUS, we noted emergence of adaptive resistance mechanisms, including upregulation of TIGIT on CD4 + T cells and CD155 on non-immune tumor and stromal cells. CONCLUSIONS: FUS BBB/BTB-D exerts mild, transient inflammatory effects in gliomas-suggesting that its combination with adjunct therapeutic strategies targeting adaptive resistance may improve outcomes. The potential for FUS-mediated BBB/BTB-D to modify immunological signatures is a timely and important consideration for ongoing clinical trials investigating this regimen in GB.

Emphysema Index Based on Hyperpolarized 3He or 129Xe Diffusion MRI: Performance and Comparison with Quantitative CT and Pulmonary Function Tests.

Background Apparent diffusion coefficient (ADC) maps of inhaled hyperpolarized gases have shown promise in the characterization of emphysema in patients with chronic obstructive pulmonary disease (COPD), yet an easily interpreted quantitative metric beyond mean and standard deviation has not been established. Purpose To introduce a quantitative framework with which to characterize emphysema burden based on hyperpolarized helium 3 (3He) and xenon 129 (129Xe) ADC maps and compare its diagnostic performance with CT-based emphysema metrics and pulmonary function tests (PFTs). Materials and Methods Twenty-seven patients with mild, moderate, or severe COPD and 13 age-matched healthy control subjects participated in this retrospective study. Participants underwent CT and multiple b value diffusion-weighted 3He and 129Xe MRI examinations and standard PFTs between August 2014 and November 2017. ADC-based emphysema index was computed separately for each gas and b value as the fraction of lung voxels with ADC values greater than in the healthy group 99th percentile. The resulting values were compared with quantitative CT results (relative lung area <-950 HU) as the reference standard. Diagnostic performance metrics included area under the receiver operating characteristic curve (AUC). Spearman rank correlations and Wilcoxon rank sum tests were performed between ADC-, CT-, and PFT-based metrics, and intraclass correlation was performed between repeated measurements. Results Thirty-six participants were evaluated (mean age, 60 years ± 6 [standard deviation]; 20 women). ADC-based emphysema index was highly repeatable (intraclass correlation coefficient > 0.99) and strongly correlated with quantitative CT (r = 0.86, P < .001 for 3He; r = 0.85, P < .001 for 129Xe) with high AUC (≥0.93; 95% confidence interval [CI]: 0.85, 1.00). ADC emphysema indices were also correlated with percentage of predicted diffusing capacity of lung for carbon monoxide (r = -0.81, P < .001 for 3He; r = -0.80, P < .001 for 129Xe) and percentage of predicted residual lung volume divided by total lung capacity (r = 0.65, P < .001 for 3He; r = 0.61, P < .001 for 129Xe). Conclusion Emphysema index based on hyperpolarized helium 3 or xenon 129 diffusion MRI provides a repeatable measure of emphysema burden, independent of gas or b value, with similar diagnostic performance as quantitative CT or pulmonary function metrics. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Schiebler and Fain in this issue.

Focused Ultrasound Preconditioning for Augmented Nanoparticle Penetration and Efficacy in the Central Nervous System.

Microbubble activation with focused ultrasound (FUS) facilitates the noninvasive and spatially-targeted delivery of systemically administered therapeutics across the blood-brain barrier (BBB). FUS also augments the penetration of nanoscale therapeutics through brain tissue; however, this secondary effect has not been leveraged. Here, 1 MHz FUS sequences that increase the volume of transfected brain tissue after convection-enhanced delivery of gene-vector "brain-penetrating" nanoparticles were first identified. Next, FUS preconditioning is applied prior to trans-BBB nanoparticle delivery, yielding up to a fivefold increase in subsequent transgene expression. Magnetic resonance imaging (MRI) analyses of tissue temperature and Ktrans confirm that augmented transfection occurs through modulation of parenchymal tissue with FUS. FUS preconditioning represents a simple and effective strategy for markedly improving the efficacy of gene vector nanoparticles in the central nervous system.

Early Identification of Autism Spectrum Disorder (ASD): Strategies for Use in Local Communities.

Early diagnosis of autism spectrum disorder (ASD) is essential for improved outcomes. There is a paucity of data on the prevalence of ASD in low- and middle-income countries (LMIC), but early identification may be further delayed in those communities. In this paper, recent studies on strategies for the early detection of ASD, and the prevalence of ASD in LMIC are reviewed. The limitations that can arise in the early identification of ASD in LMIC communities are discussed, and screening tools and strategies that can be helpful are identified. The goal is to recommend models that are culturally appropriate and scientifically valid, easily integrated within community settings while strengthening community systems and reducing disparities in the early identification of ASD. Starting locally by simplifying and demystifying the ASD identification process and building community connections will inform global researchers and policymakers while making a difference in the lives of the children and families affected by ASD.

The RITA-T (Rapid Interactive Screening Test for Autism in Toddlers) Community Model to Improve Access and Early Identification of Autism in Young Children.

Objective: To evaluate improved identification and the generalization of the RITA-T (Rapid interactive Screening Test for Autism in Toddlers) model through partnerships with Primary Care (PC), Early Intervention (EI), and Autism Diagnosticians. Methods: Over 3 years (2018-2021), 15 EI and 9 PC (MD and NP) centers participated in this project. We trained providers on the RITA-T and established screening models. We reviewed charts of all toddlers referred through this model and compared wait times, and diagnoses, to those evaluated through regular referral in a tertiary-based autism clinic. We also examined the RITA-T psychometrics. Results: 377 toddlers met our inclusion criteria. Wait time for diagnosis was an average of 2.8 months and led to further collaboration between community providers. RITA-T cut-off scores stayed consistent. Providers reported improved confidence and easy integration of this model. Conclusions: This model is generalizable and improves the Early Identification of ASD.

Acquiring Hyperpolarized 129Xe Magnetic Resonance Images of Lung Ventilation.

Hyperpolarized 129Xe MRI comprises a unique array of structural and functional lung imaging techniques. Technique standardization across sites is increasingly important given the recent FDA approval of 129Xe as an MR contrast agent and as interest in 129Xe MRI increases among research and clinical institutions. Members of the 129Xe MRI Clinical Trials Consortium (Xe MRI CTC) have agreed upon best practices for each of the key aspects of the 129Xe MRI workflow, and these recommendations are summarized in a recent publication. This work provides practical information to develop an end-to-end workflow for collecting 129Xe MR images of lung ventilation according to the Xe MRI CTC recommendations. Preparation and administration of 129Xe for MR studies will be discussed and demonstrated, with specific topics including choice of appropriate gas volumes for entire studies and for individual MR scans, preparation and delivery of individual 129Xe doses, and best practices for monitoring subject safety and 129Xe tolerability during studies. Key MR technical considerations will also be covered, including pulse sequence types and optimized parameters, calibration of 129Xe flip angle and center frequency, and 129Xe MRI ventilation image analysis.

Lung Volume Dependence and Repeatability of Hyperpolarized 129Xe MRI Gas Uptake Metrics in Healthy Volunteers and Participants with COPD.

Purpose: To assess the effect of lung volume on measured values and repeatability of xenon 129 (129Xe) gas uptake metrics in healthy volunteers and participants with chronic obstructive pulmonary disease (COPD). Materials and Methods: This Health Insurance Portability and Accountability Act-compliant prospective study included data (March 2014-December 2015) from 49 participants (19 with COPD [mean age, 67 years ± 9 (SD)]; nine women]; 25 older healthy volunteers [mean age, 59 years ± 10; 20 women]; and five young healthy women [mean age, 23 years ± 3]). Thirty-two participants underwent repeated 129Xe and same-breath-hold proton MRI at residual volume plus one-third forced vital capacity (RV+FVC/3), with 29 also undergoing one examination at total lung capacity (TLC). The remaining 17 participants underwent imaging at TLC, RV+FVC/3, and residual volume (RV). Signal ratios between membrane, red blood cell (RBC), and gas-phase compartments were calculated using hierarchical iterative decomposition of water and fat with echo asymmetry and least-squares estimation (ie, IDEAL). Repeatability was assessed using coefficient of variation and intraclass correlation coefficient, and volume relationships were assessed using Spearman correlation and Wilcoxon rank sum tests. Results: Gas uptake metrics were repeatable at RV+FVC/3 (intraclass correlation coefficient = 0.88 for membrane/gas; 0.71 for RBC/gas, and 0.88 for RBC/membrane). Relative ratio changes were highly correlated with relative volume changes for membrane/gas (r = -0.97) and RBC/gas (r = -0.93). Membrane/gas and RBC/gas measured at RV+FVC/3 were significantly lower in the COPD group than the corresponding healthy group (P ≤ .001). However, these differences lessened upon correction for individual volume differences (P = .23 for membrane/gas; P = .09 for RBC/gas). Conclusion: Dissolved-phase 129Xe MRI-derived gas uptake metrics were repeatable but highly dependent on lung volume during measurement.Keywords: Blood-Air Barrier, MRI, Chronic Obstructive Pulmonary Disease, Pulmonary Gas Exchange, Xenon Supplemental material is available for this article © RSNA, 2023.

A replicon RNA vaccine induces durable protective immunity from SARS-CoV-2 in nonhuman primates after neutralizing antibodies have waned.

The global SARS-CoV-2 pandemic prompted rapid development of COVID-19 vaccines. Although several vaccines have received emergency approval through various public health agencies, the SARS-CoV-2 pandemic continues. Emergent variants of concern, waning immunity in the vaccinated, evidence that vaccines may not prevent transmission and inequity in vaccine distribution have driven continued development of vaccines against SARS-CoV-2 to address these public health needs. In this report, we evaluated a novel self-amplifying replicon RNA vaccine against SARS-CoV-2 in a pigtail macaque model of COVID-19 disease. We found that this vaccine elicited strong binding and neutralizing antibody responses. While binding antibody responses were sustained, neutralizing antibody waned to undetectable levels after six months but were rapidly recalled and conferred protection from disease when the animals were challenged 7 months after vaccination as evident by reduced viral replication and pathology in the lower respiratory tract, reduced viral shedding in the nasal cavity and lower concentrations of pro-inflammatory cytokines in the lung. Cumulatively, our data demonstrate in pigtail macaques that a self-amplifying replicon RNA vaccine can elicit durable and protective immunity to SARS-CoV-2 infection. Furthermore, these data provide evidence that this vaccine can provide durable protective efficacy and reduce viral shedding even after neutralizing antibody responses have waned to undetectable levels.

Improving Early Identification and Access to Diagnosis of Autism Spectrum Disorder in Toddlers in a Culturally Diverse Community with the Rapid Interactive screening Test for Autism in Toddlers.

The objective of this study was to test a screening model that employs the Rapid Interactive Screening Test for Autism in Toddlers (RITA-T), in an underserved community to improve ASD detection. We collaborated with a large Early Intervention (EI) program and trained 4 providers reliably on the RITA-T. Toddlers received the Modified Checklist for Autism in Toddlers (MCHAT-R/F), the RITA-T, developmental and autism testing, and a best-estimate clinical diagnosis. Eighty-One toddlers were enrolled: 57 with ASD and 24 with Developmental Delay (DD) non-ASD. Wait-time for diagnosis was on average 6 weeks. The RITA-T correlated highly with autism measures and EI staff integrated this model easily. The RITA-T significantly improved the identification and wait time for ASD in this underserved community.

A Single Dose SARS-CoV-2 Replicon RNA Vaccine Induces Cellular and Humoral Immune Responses in Simian Immunodeficiency Virus Infected and Uninfected Pigtail Macaques.

The ongoing COVID-19 vaccine rollout is critical for reducing SARS-CoV-2 infections, hospitalizations, and deaths worldwide. Unfortunately, massive disparities exist in getting vaccines to vulnerable populations, including people living with HIV. Preliminary studies indicate that COVID-19 mRNA vaccines are safe and immunogenic in people living with HIV that are virally suppressed with potent antiretroviral therapy but may be less efficacious in immunocompromised individuals. This raises the concern that COVID-19 vaccines may be less effective in resource poor settings with limited access to antiretroviral therapy. Here, we evaluated the immunogenicity of a single dose COVID-19 replicon RNA vaccine expressing Spike protein (A.1) from SARS-CoV-2 (repRNA-CoV2S) in immunocompromised, SIV infected and immune competent, naïve pigtail macaques. Moderate vaccine-specific cellular Th1 T-cell responses and binding and neutralizing antibodies were induced by repRNA-CoV2S in SIV infected animals and naïve animals. Furthermore, vaccine immunogenicity was elicited even among the animals with the highest SIV viral burden or lowest peripheral CD4 counts prior to immunization. This study provides evidence that a SARS-CoV-2 repRNA vaccine could be employed to induce strong immunity against COVID-19 in HIV infected and other immunocompromised individuals.

Transcriptomic response of brain tissue to focused ultrasound-mediated blood-brain barrier disruption depends strongly on anesthesia.

Focused ultrasound (FUS) mediated blood-brain barrier disruption (BBBD) targets the delivery of systemically-administered therapeutics to the central nervous system. Preclinical investigations of BBBD have been performed on different anesthetic backgrounds; however, the influence of the choice of anesthetic on the molecular response to BBBD is unknown, despite its potential to critically affect interpretation of experimental therapeutic outcomes. Here, using bulk RNA sequencing, we comprehensively examined the transcriptomic response of both normal brain tissue and brain tissue exposed to FUS-induced BBBD in mice anesthetized with either isoflurane with medical air (Iso) or ketamine/dexmedetomidine (KD). In normal murine brain tissue, Iso alone elicited minimal differential gene expression (DGE) and repressed pathways associated with neuronal signaling. KD alone, however, led to massive DGE and enrichment of pathways associated with protein synthesis. In brain tissue exposed to BBBD (1 MHz, 0.5 Hz pulse repetition frequency, 0.4 MPa peak-negative pressure), we systematically evaluated the relative effects of anesthesia, microbubbles, and FUS on the transcriptome. Of particular interest, we observed that gene sets associated with sterile inflammatory responses and cell-cell junctional activity were induced by BBBD, regardless of the choice of anesthesia. Meanwhile, gene sets associated with metabolism, platelet activity, tissue repair, and signaling pathways, were differentially affected by BBBD, with a strong dependence on the anesthetic. We conclude that the underlying transcriptomic response to FUS-mediated BBBD may be powerfully influenced by anesthesia. These findings raise considerations for the translation of FUS-BBBD delivery approaches that impact, in particular, metabolism, tissue repair, and intracellular signaling.

Immunomodulation of intracranial melanoma in response to blood-tumor barrier opening with focused ultrasound.

Background: Focused ultrasound (FUS) activation of microbubbles (MBs) for blood-brain (BBB) and blood-tumor barrier (BTB) opening permits targeted therapeutic delivery. While the effects of FUS+MBs mediated BBB opening have been investigated for normal brain tissue, no such studies exist for intracranial tumors. As this technology advances into clinical immunotherapy trials, it will be crucial to understand how FUS+MBs modulates the tumor immune microenvironment. Methods and Results: Bulk RNA sequencing revealed that FUS+MBs BTB/BBB opening (1 MHz, 0.5 MPa peak-negative pressure) of intracranial B16F1cOVA tumors increases the expression of genes related to proinflammatory cytokine and chemokine signaling, pattern recognition receptor signaling, and antigen processing and presentation. Flow cytometry revealed increased maturation (i.e. CD86) of dendritic cells (DCs) in the meninges and altered antigen loading of DCs in both the tumor and meninges. For DCs in tumor draining lymph nodes, FUS+MBs had no effect on maturation and elicited only a trend towards increased presentation of tumor-derived peptide by MHC. Neither tumor endothelial cell adhesion molecule expression nor homing of activated T cells was affected by FUS+MBs. Conclusion: FUS+MBs-mediated BTB/BBB opening elicits signatures of inflammation; however, the response is mild, transient, and unlikely to elicit a systemic response independent of administration of immune adjuvants.

Augmentation of brain tumor interstitial flow via focused ultrasound promotes brain-penetrating nanoparticle dispersion and transfection.

The delivery of systemically administered gene therapies to brain tumors is exceptionally difficult because of the blood-brain barrier (BBB) and blood-tumor barrier (BTB). In addition, the adhesive and nanoporous tumor extracellular matrix hinders therapeutic dispersion. We first developed the use of magnetic resonance image (MRI)-guided focused ultrasound (FUS) and microbubbles as a platform approach for transfecting brain tumors by targeting the delivery of systemically administered "brain-penetrating" nanoparticle (BPN) gene vectors across the BTB/BBB. Next, using an MRI-based transport analysis, we determined that after FUS-mediated BTB/BBB opening, mean interstitial flow velocity magnitude doubled, with "per voxel" flow directions changing by an average of ~70° to 80°. Last, we observed that FUS-mediated BTB/BBB opening increased the dispersion of directly injected BPNs through tumor tissue by >100%. We conclude that FUS-mediated BTB/BBB opening yields markedly augmented interstitial tumor flow that, in turn, plays a critical role in enhancing BPN transport through tumor tissue.