Sensitizing breast cancers to immune checkpoint inhibitors through CD27 agonism and vaccination against tumor-associated antigen

Project objective: Despite the recent revolution in immune checkpoint inhibitors (ICIs), only modest improvement in overall survival and likely caused by not enough potent cellular immunity among BC patients. Our lab has been focus on inducing cellular immunity against HER2+ BC through vaccination against the tumor-associated antigen HER2. Approximately 20 years ago, we performed an experimental pilot study by administrating HER2 peptide and recombinant protein pulsed dendritic cells (DC vaccine) to six patients with refractory HER2+ advanced or metastatic (stage II (>= 6 +LN), III, or stage IV) BC. We followed the patients on 2019 found that all of the six patients were still alive, 18 years after vaccination. Their blood sample were analyzed with cytometry by time-offlight (CyTOF) and found there is a significantly increased presence of CD27 expressing memory T cells in response to HER2 peptide stimulation. Recent report on the SARS-CoV2 mRNA vaccine also suggested that CD27 expressing memory T cells plays a critical role in long-lasting cellular immunity against SARS-CoV2 infection. Therefore, we hypothesized that CD27 plays a critical role in cellular immunity against BC, and the stimulation of CD27 expressing T cells with mAb targeting CD27 significantly increase the cellular immunity triggered by vaccination against tumor-associated antigen. Result(s): We recapitulate the rise of CD27+ antigen specific T cells among the vaccinated patients using a transgenic mouse model expressing human CD27. When combined the adenoviral-vector based HER2 (Ad-HER2) vaccination with a single dose of human aCD27 antibody (Varlilumab), we found there is a robust increase in the HER2 specific T cells compared to vaccination alone, especially CD27+CD44+ memory CD4 T cells, even after 120 days post vaccination. Using an ICIinsensitive syngeneic HER2+ BC models, we found 50% of mice in the combination group of aCD27 antibody plus Ad-HER2 showed total tumor regression by the end of study. When combined with anti-PD1 antibody, the combination of AdHER2 and Varlilumab leads to total tumor regression in 90% of tumor bearing mice with syngeneic HER2+ BC, indicating that the vaccination against tumor associated antigen HER2 plus anti-CD27 antibody sensitized ICI-insensitive HER2+ BC toward ICI. Conclusion(s): Our data demonstrates that the administration of anti-CD27 antibody significantly increase the long term presence of CD27+ antigen specific memory T cells after vaccination against tumor associated antigen HER2. As consequence, combination of anti-CD27 with HER2 sensitized the immune unresponsive breast cancer toward anti-PD1 antibody. Our study suggests that the vaccination against tumor-associated antigen with mAb targeting CD27 leads to the robust cellular immunity, which is required for successful ICIs against breast cancer.

Highly synergistic combinations of nanobodies that target SARS-CoV-2 reduce viral load in hACE2 transgenic mice

The ongoing emergence of SARS-CoV-2 variants threatens current vaccines and renders current therapeutic antibodies obsolete, demanding powerful new treatments that can resist viral escape. We therefore generated a large nanobody repertoire to saturate the distinct and highly conserved available epitope space of SARS-CoV-2 spike, including the S1 receptor binding domain, N-terminal domain, and the S2 subunit, to identify new nanobody binding sites that may reflect novel mechanisms of viral neutralization. Structural mapping and functional assays show that these highly stable monovalent nanobodies potently inhibit SARS-CoV-2 infection, display numerous neutralization mechanisms, are effective against past and present emerging variants of concern, and are resistant to mutational escape. Rational combinations of these nanobodies that bind dissimilar sites within and between spike subunits exhibit extraordinary synergy and suggest multiple tailored therapeutic and prophylactic strategies. All mouse involved experiments were performed in compliance with the Institutional Animal Care and Use Committee and mice were housed and maintained in a specific pathogen-free conditions at Seattle Children's Research Institute. Infected mice with SARSCoV- 2 were housed in a Biosafety Level 3 facility in an Animal Biohazard Containment Suite. Prophylactic intranasal application of a synergistic pair of unmodified nanobodies in 10-12 week-old female K18-hACE2 transgenic mice, a mouse model of SARS-CoV-2 infection, showed significant reduction in viral load after 3 days post-challenge with SARS-CoV-2, the first demonstration of synergy in vivo. In summary, our results show that our diverse repertoire of nanobodies can neutralize current variants of live SARS-CoV-2, pairs of nanobodies that bind distinct sites on spike show tremendous synergy in neutralizing efficacy in vitro, and the application of synergizing pair of nanobodies translates to an in vivo mouse model of SARSCoV- 2. Research funded by the Mathers Foundation, Robertson Foundation, NIH P41GM109824.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

SARS-CoV-2 E-PROTEIN VIROPORIN INHIBITOR BIT225 ACTIVE IN hACE2 TRANSGENIC MICE

Background: The CoV-2 envelope (E) protein plays an important role in virus assembly, budding, immunopathogenesis and disease severity. E protein has ion channel activity, is located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we report that BIT225, an investigational HIV clinical compound, inhibits E ion channel activity and prevents body weight loss and mortality and reduces inflammation in lethally infected K18-hACE2 transgenic mice. BIT225 efficacy was observed when dosing was initiated before or 24 h or 48 h after infection. Method(s): SARS-CoV-2 E protein ion channel activity and Xenopus TMEM16A were measured in Xenopus oocytes. K18-hACE2 transgenic mice were infected intranasally with 104 pfu SARS CoV 2 (US-WA1/2020) and dosed orally twice daily with BIT225 for up to 12 Days. Dosing was initiated 12 h pre-infection or 24 h or 48 h post-infection. Disease parameters measured were survival, body weight, viral RNA by qPCR and infectious virus titre (plaque assay) in lung tissue homogenates and serum. In addition, levels of pro-inflammatory cytokines (IL-6, IL-1alpha, IL-1beta, TNFalpha & TGFbeta, MCP-1) were measured in lung and serum samples. Result(s): BIT225 inhibited ion channel activity of E-protein, but not that of TMEM16A in Xenopus oocytes. BIT225 dosed at 300mg/kg BID for 12 days starting 12 h pre-infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (n=12), while all vehicle-dosed animals reached a mortality endpoint by day 9 across two studies (n=12). Figure 1 shows results from a time of addition study: When treatment with BIT225 started at 24 h post-infection, body weight loss and mortality was also prevented (100% survival, n=5). In the group of mice where treatment started at 48 h after infection, body weight loss and mortality were prevented in 4 of 5 mice. Treatment efficacy was associated with significant reduction in lung viral load (3.5 log10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. Conclusion(s): BIT225 is an inhibitor of SARS-CoV-2 E-protein viroporin activity. In the K18 model BIT225 protected mice from weight loss and death, inhibited virus replication and reduced inflammation. These effects were noted when treatment with BIT225 was initiated before or 24-48 hours after infection and validate viroporin E as a viable antiviral target and support the clinical study of BIT225 in treatment of SARS-CoV-2.

Effect of severe acute respiratory syndrome coronavirus 2 infection during pregnancy in K18-hACE2 transgenic mice.

Objective: This study aimed to examine the influence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on pregnancy in cytokeratin-18 (K18)-hACE2 transgenic mice. Methods: To determine the expression of hACE2 mRNA in the female reproductive tract of K18-hACE2 mice, RT-PCR was performed using the ovary, oviduct, uterus, umbilical cord, and placenta. SARS-CoV-2 was inoculated intranasally (30 µl/mouse, 1×104 TCID50/ml) to plug-checked K18-hACE2 homozygous female mice at the pre-and post-implantation stages at 2.5 days post-coitum (dpc) and 15.5 dpc, respectively. The number of implantation sites was checked at 7.5 dpc, and the number of normally born pups was investigated at 20.5 dpc. Pregnancy outcomes, including implantation and childbirth, were confirmed by comparison with the non-infected group. Tissues of infected mice were collected at 7.5 dpc and 19.5 dpc to confirm the SARS-CoV-2 infection. The infection was identified by performing RT-PCR on the infected tissues and comparing them to the non-infected tissues. Results: hACE2 mRNA expression was confirmed in the female reproductive tract of the K18-hACE2 mice. Compared to the non-infected group, no significant difference in the number of implantation sites or normally born pups was found in the infected group. SARS-CoV-2 infection was detected in the lungs but not in the female reproductive system of infected K18-hACE2 mice. Conclusion: In K18-hACE2 mice, intranasal infection with SARS-CoV-2 did not induce implantation failure, preterm labor, or miscarriage. Although the viral infection was not detected in the uterus, placenta, or fetus, the infection of the lungs could induce problems in the reproductive system. However, lung infections were not related to pregnancy outcomes.

Middle East Respiratory Syndrome-Coronavirus Infection into Established hDPP4-Transgenic Mice Accelerates Lung Damage Via Activation of the Pro-Inflammatory Response and Pulmonary Fibrosis.

Middle East respiratory syndrome coronavirus (MERS-CoV) infects the lower respiratory airway of humans, leading to severe acute respiratory failure. Unlike human dipeptidyl peptidase 4 (hDPP4), a receptor for MERS-CoV, mouse DPP4 (mDPP4) failed to support MERS-CoV infection. Consequently, diverse transgenic mouse models expressing hDPP4 have been developed using diverse methods, although some models show no mortality and/or only transient and mild-to-moderate clinical signs following MERS-CoV infection. Additionally, overexpressed hDPP4 is associated with neurological complications and breeding difficulties in some transgenic mice, resulting in impeding further studies. Here, we generated stable hDPP4-transgenic mice that were sufficiently susceptible to MERS-CoV infection. The transgenic mice showed weight loss, decreased pulmonary function, and increased mortality with minimal perturbation of overexpressed hDPP4 after MERS-CoV infection. In addition, we observed histopathological signs indicative of progressive pulmonary fibrosis, including thickened alveolar septa, infiltration of inflammatory monocytes, and macrophage polarization as well as elevated expression of profibrotic molecules and acute inflammatory response in the lung of MERS-CoV-infected hDPP4-transgenic mice. Collectively, we suggest that this hDPP4-transgenic mouse is useful in understanding the pathogenesis of MERS-CoV infection and for antiviral research and vaccine development against the virus.

Microbial Dysbiosis and Epithelial Dysfunction in Vitamin A-Deficient Lungs

Once believed to be sterile, recent studies now show microbes inhabiting healthy lungs that are dysregulated in patients with chronic obstructive pulmonary disease (COPD), asthma, tuberculosis (TB), and SARS-CoV-2 infection. Other studies have shown an increase in pulmonary disease and recurrent respiratory infections in malnourished patients. According to the World Health Organization, vitamin A deficiency (VAD) is now a major public health issue in low-income communities and many developing countries. While VAD has been shown to alter gene expression and tissue morphology in humans and mice, research suggests the lung microbiome plays an intimate role in the metabolic regulation, pathogen inhibition, and inflammatory responses in the lung. Whether dysbiosis is a cause or consequence of chronic respiratory conditions, or whether retinoic acid (RA) - the bioactive metabolite of Vitamin A - is essential for lung microbiome homeostasis, remains unknown. Therefore, we hypothesize that dietary VAD leads to epithelial remodeling which promotes microbial dysbiosis;the dysbiosis then perpetuates epithelial remodeling via host-microbe interactions. Our preliminary results show anatomical/pathological changes to the epithelium in VAD adult mouse lungs compared to controls (VAS). Using our Nkx2- 1creERT2/dnRAR Rosa26 tdTomato transgenic mouse model that selectively induces VAD in the adult lung epithelium following tamoxifen injections, our data supports the hypothesis that host epithelial aberration associated with dietary VAD is induced locally in the lung and not via distal or systemic mechanisms. Our data also indicates the onset of dysbiosis in adult mouse lungs as early as three weeks post-diet modulation as observed through changes in microbial composition in VAD mice compared to controls. Finally, our bulk RNAseq analysis of host and microbial gene signatures has uncovered mechanisms associated with microbial metabolic functions, ciliopathy, host cellular polarity, and immune response to infection, that are dysregulated in the absence of vitamin A. Further, we have also identified altered transcriptional activity of microbes that are traditionally symbiotic or pathobiotic under normal homeostasis. This work indicates the presence of specific host-microbe interactions that are essential for lung homeostasis and protection against lung infection and disease that are dysregulated or lost in the absence of dietary vitamin A.

GALECTIN-9 PROTECTS HUMANIZED ACE2-IMMUNOCOMPETENT MICE from SARS-CoV-2 INFECTION

Background: Coronavirus disease 2019 (COVID19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) remains a global health emergency even with effective vaccines and limited FDA-approved therapies. To limit mortality and morbidity across the spectrum of disease, the need for therapeutics remains critical. Galectin9 (gal9) is a beta-galactoside binding protein that modulates cell-cell and cell-matrix interactions. In response to SARS-CoV2 infection, it has been shown that circulating gal9 levels are elevated in patient sera with moderate to severe disease. Additionally, it has been reported that gal9 unexpectedly may competitively bind the host ACE2 receptor, potentially impeding viral entry. Therefore, we hypothesized that early recombinant gal-9 treatment post infection may prevent binding of the virus to susceptible host cells resulting in decreased severity of SARS-CoV2-associated disease. Methods: To determine the therapeutic potential of gal9 for treating COVID19, we infected K18-hACE2 transgenic mice intranasally with 104 particle forming units (PFU) of SARS-CoV2. 6 hours post infection (hpi), mice were treated with a single dose of 30 ug of recombinant human gal9 (rhgal9) or PBS intraperitoneally and subsequently monitored 12 days for morbidity. Subgroups of mice were humanely euthanized at 2 and 5 days pi (dpi) for viral plaque assay, flow cytometry, and protein analysis from lung tissue and bronchial alveolar lavage (BAL). Results: We found that mice treated with rhgal9 during the acute phase of infection exhibit improved survival compared to PBS treated animals (25%, p<0.0001). We found that at 5 dpi, rhgal9 treated mice exhibited enhanced viral clearance in the BAL but not in the lung parenchyma. Additionally, we found increased CD8 T cell (p<0.001) and decreased neutrophil (p<0.05) frequencies in the lung at 5 dpi. Finally, we found that BAL fluid had elevated levels of Type 1 Interferon [IFNa (p<0.01) and IFNb (p<0.01)] at 2 dpi and increased MyD88 proinflammatory cytokines [IL1a (p<0.05), IL1b (p<0.01), TNFa (p<0.05), and MIP1a (p<0.05) at 5 dpi. Conclusion: Our study suggests that rhgal9 treatment may be potentially therapeutic for treating acute COVID19. Our data suggest that rhgal9 treatment in combination with other anti-inflammatory mediators may curtail damaging inflammation associated with SARS-CoV2 disease. Further studies are required to determine the optimal time, combination and duration of treatment pi to effectively target the gal9 pathways.

LONG-TERM IMMUNOGENICITY and EFFECTIVENESS of CD40-TARGETING VACCINATION in COVID-19

Background: Following natural infection or vaccination, the generation of stem cell-like memory T (Tscm) cells is essential for long-term protective immunity to the virus. Tscm cells have the capacity for self-renewal and multipotency. In SARS-CoV2 infection, the emergence of CD8+ Tscm cells is correlated with the number of symptom-free days. The development of a COVID-19 vaccine able to generate CD8+ Tscm cells is of the utmost importance since the emergence of SARS-CoV2 variants of concerns requires maintaining strong and long-lasting immune responses, 2) as an efficient alternative in immunocompromised people who have difficulties raising humoral immune responses. Methods: We have developed a new Dendritic Cell-based vaccine composed of a humanized αCD40 monoclonal antibody fused to the RBD protein in its C-terminal Fc-domains and three T cell epitopes spanning sequences from S and N proteins in its light chains (αCD40-CoV2). Previous studies have shown that this platform elicited durable and robust T-and B-cell responses and is currently in phase I clinical development in HIV. We tested the capacity of two injections of the vaccine (10υg, i.p) given with or without poly(IC) (50υg, i.p) at 3 weeks apart to i) elicit human (hu) B-, and huT-cell responses in NSG mice reconstituted with a Human Immune System (HIS mice), ii) protect against SARS-CoV2 infection in the hCD40xK18hACE2 transgenic mice. Results: We performed AIM assays and intracellular staining on spleen cells of HIS mice stimulated with overlapping peptide pools spanning the sequences of vaccine antigens. We found that both non-adjuvanted and adjuvanted vaccine efficiently induced SARS-CoV2-specific Th1 huCD4+ and huCD8+ T cells in all vaccinees compared to mock animals. SARS-CoV2-specific huCD4+ T cells were polyfunctional. We confirmed the presence of RBD-specific huCD8+ T cells in the vaccinated animals using HLA-I tetramers. A significant proportion of the multimer+ huCD8+ T cells were Tscm (CD45RA+ CD62L+ CD95+) cells in both vaccinated groups. Besides, we detected significant amounts of spike-IgG+ switched huB cells in all vaccinees. In SARS-CoV2 challenge experiments, we further showed that both vaccination settings significantly protected animals with a survival rate of 100%. Conclusion: We demonstrate that the targeting of SARS-CoV-2 epitopes to CD40 induces significant B and T cells with a long-term memory phenotype in HIS mice and the ability of the vaccine to ensure complete protection against SARS-CoV2 infection.

IN VIVO EFFICACY of ENGINEERED ACE2-Fc in PREVENTING LETHAL SARS-CoV-2 INFECTION

Background: Soluble Angiotensin Converting Enzyme 2 (ACE2) constitutes an attractive therapeutic candidate with natural resistance to viral escape. To date, ACE2-Fcs, dimeric forms of soluble ACE2, were mostly tested as robust SARS-CoV-2 neutralizers but their potential as antiviral agents capable of Fc-effector functions is largely unknown and has not been tested for effectiveness in vivo, in any model of SARS-CoV2 infection. Methods: We used structure-guided design to select ACE2 mutations that improve SARS-CoV-2 spike (S) affinity and remove angiotensin enzymatic activity. ACE2-Fc variants were engineered into a human IgG1 or IgG3 backbone and produced in mammalian HEK293 cells. S binding was tested by ELISA and surface plasmon resonance (SPR). Mutational effects were validated by X-Ray crystallography. Neutralization activities were measured against SARS-CoV-2 variants of concern (VOCs) using an in vitro pseudovirus (PsV) assay and dynamic bioluminescence imaging (BLI). Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) were also quantified using established methods (1, 2). A K18-hACE2 transgenic mouse model challenged by lethal SARS-CoV-2 nLuc infection (3) was used for in vivo evaluation of prophylactic and therapeutic administration of engineered ACE2-Fcs, as monitored by dynamic BLI. Results: Our lead variant, ACE2740 LFMYQY2HA-Fc GASDALIE, increased RBD binding by ∼7-13 fold as compared to wild type, cross-neutralized SARS-CoV-2 VOCs with an IC50 range of 0.23-2.06 nM and mediated robust ADCC and ADCP in vitro. When tested in humanized K18-hACE2 mice, in either a prophylatic or a multi-dosage therapeutic setting, our lead ACE2-Fc variant provided protection from lethal SARS-CoV-2 infection. Our studies in K18-hACE2 mouse model revealed that efficient in vivo efficacy of ACE2-Fcs under prophylaxis or therapeutic settings required Fc-effector functions in addition to neutralization. Conclusion: Our data confirm the utility of engineered ACE2-Fcs as valuable SARS-CoV-2 antivirals and demonstrate that the efficient ACE2-Fc therapeutic activity required both neutralization and Fc-effector functions.

A FC-ENHANCED NON-NEUTRALIZING ANTIBODY DELAYS SARS-CoV-2-INDUCED DEATH in MICE

Background: Both neutralizing activity and Fc-mediated effector functions of antibodies are believed to contribute to protection against SARS-CoV-2. However, it is unclear if antibody effector functions alone could protect against SARS-CoV-2 infection. Methods: We isolated CV3-13 from a convalescent individual with potent Fc-mediated effector functions. Neutralization capacity of this antibody was measured by both a pseudovirus neutralization assay and an authentic virus microneutralization assay. We mutated the Fc-portion of CV3-13 to enhance (GASDALIE) or reduce (LALA) its capacity to mediate antibody dependant cellular cytotoxicity (ADCC). Structural analysis of CV3-13 was done by cryo-EM to characterize its epitope and its angle of approach. Finally, CV3-13 and CV3-13 GASDALIE were used in vivo in a K18-hACE2 transgenic mouse model challenged with SARS-CoV-2-nLuc to see if they altered viral replication and/or contributed to protection against SARS-CoV-2. Results: While CV3-13 did not neutralize SARS-CoV-2, it demonstrated nanomolar affinity towards the SARS-CoV-2 Spike and mediated strong ADCC. The cryo-EM structure of CV3-13 in complex with the SARS-CoV-2 Spike revealed that the antibody bound to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. Interestingly, this angle of approach was not observed for previously described NTD-directed antibodies. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, a Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Conclusion: CV3-13 represents a new class of non-neutralizing NTD-directed mAbs that can mediate Fc-effector functions both in vitro and in vivo. While effector functions alone did not protect K18-hACE2 mice from SARS-CoV-2-nLuc challenge, our data indicate that along with neutralization, additional antibody properties including Fc-mediated effector functions contribute to limiting viral spread and aid in fighting SARS-CoV-2 infection.

Histopathologic Pulmonary Involvement of COVID-19: A Comparative Study of SARS-CoV-2 Variants in Mouse Models

Background: A thorough understanding of the inflammatory reaction to SARS-CoV-2 variants, specifically the Delta and Alpha variants, can provide crucial insight into future treatment of individuals infected with these strains. Mice are an effective model for predicting the pathologic processes of these viruses in humans. Design: K18-hACE2 transgenic mice were raised either under normal conditions (control) or infected with either the Alpha (strain B.1.1.7) or Delta (B.1.617.2) variant of SARS-CoV-2, via intranasal challenge with 1000 PFU virus. Mice were then euthanized at days 2 and 6 post-challenge. Lung sections were used for pathological evaluation of H&E stained slides scanned using the Dynamyx software. Blood vessel cross sections were examined and the average number of marginating inflammatory cells per millimeter of vessel were quantified. Additionally, the percentage of total tissue area that was inflamed was calculated. Results: Our data indicates that the Delta variant elicits a strong lymphocytic immune response in the lungs. At two days postchallenge, Inflammation and margination of inflammatory cells could be appreciated in Delta infected mice, but not in Alpha infected mice. The inflammatory infiltrate was composed predominantly of lymphocytes and occasional histiocytes at 2 days. By day 6, marked perivascular inflammation and margination was appreciated, more prominently in Delta (36 lymphocytes per mm. of endothelium) than Alpha infected mice (22 lymphocytes per mm. endothelium) (p=0.022). At this time point, Alpha infected mice showed 4% involvement of pulmonary area by inflammation, compared to 20% for Delta infected mice (p=0.014). Conclusions: This study quantifies the lymphocytic immune response in the lungs to the Alpha and Delta variants of SARS-CoV-2 in mouse models. Both variants showed a lymphocyte predominant inflammatory response. However, the response was much more robust and severe in Delta infected mice compared to Alpha. The results support why the Delta variant is more virulent and fatal compared to Alpha. Ongoing longer term studies and effects in other organs are ongoing and will help to provide further insight into pathogenesis of long COVID-19.

MACHINE LEARNING AND RADIOMICS IDENTIFY NOVEL BIOMARKERS OF BONE LOSS

Purpose: Altered bone turnover is a factor in many diseases including osteoarthritis (OA), osteoporosis, inflammation, and viral infection. The absence of obvious symptoms and insufficiently sensitive biomarkers in the early stages of bone loss limits early diagnosis and treatment. Therefore, it is urgent to identify novel, more sensitive, and easy-to-detect biomarkers which can be used in the diagnosis and prognosis of bone health. Our previous data using standard micro-computed tomography (μCT) measurements showed that SARS-CoV-2 infection in mice significantly decreased trabecular bone volume at the lumbar spine, suggesting that decreased bone mass, increased fracture risk, and OA may be underappreciated long-haul comorbidities for COVID patients. In this study, we applied integrated state-of-the-art radiomics and machine learning models to identify more sensitive image-based biomarkers of SARS-CoV-2-induced bone loss from μCT images. These radiomic biomarkers can potentially provide a non-invasive way of quantifying and monitoring systemic bone loss and evaluating treatment efficacy in both research and clinical practices. Methods: All animal use was performed with approval of the Institutional Animal Care and Use Committee. To quantify SARS-CoV-2-induced bone loss, 6-week-old transgenic mice (16 male, 16 female) expressing humanized ACE2 receptors were inoculated with a 2020 strain of SARS-CoV-2 or phosphate-buffered saline (Control) [Fig. A]. Viral infection was confirmed by detection of infectious SARS-CoV-2 in throat swabs and histological identification of SARS-CoV-2 labeled cells. At 6-14 days post-infection, lumbar vertebral bodies (L5) were scanned with μCT (μCT 35, SCANCO Medical AG;6 μm nominal voxel size). The open-source research platform 3D Slicer v2020 with a built-in Python console v3.8 was used for medical image computing and fully automated segmentation of cortical and trabecular bone. Standard μCT assessment of bone microstructure was performed. Radiomic feature extraction and data processing were performed using python based PyRadiomics v3.0.1. A total of 120 radiographic features were extracted from the segmented images [Fig. B]. Principle component analysis (PCA) for feature selection, a support vector machine learning (SVML) predictive model for classification, holdback method for model validation, and all statistical analyses (significance at p<0.05) were performed using JMP Pro v15 (SAS). Results: Using standard μCT methods, SARS-CoV-2 infection significantly reduced the bone volume fraction (BV/TV) by 10 and 10.5% (p= 0.04) and trabecular thickness (Tb.Th) by 8 and 9% (p= 0.02) in male and female mice, respectively, compared to PBS control mice [Fig. C]. Radiomics detected a 20-fold greater magnitude in change over standard methods. SARS-CoV-2 infection significantly changed radiographic parameters with the largest change being a 300% increase in the second-order parameter: cluster shade [Fig. D]. The 45 radiomic features comprising the first 3 principal components were selected for inclusion in the SVML model. The SVML Model (radial basis function kernel;cost = 4.8;gamma = 0.46) produced an area under the receiver operating characteristic curve (AUC) of 1.0 which reflects a perfectly accurate test [Fig. E]. Conclusions: SARS-CoV-2 infection of humanized ACE2 expressing mice caused significant bone changes, suggesting that decreased bone mass, increased fracture risk, OA, and other musculoskeletal complications could be long-term comorbidities for people infected with COVID-19. We developed an open-source, fully automated segmentation and radiomics system to assess systemic bone loss using μCT images. When coupled with machine learning, this system was able to identify novel radiographic biomarkers of bone loss that better discriminate differences in bone microstructure between SARS-CoV-2 infected and control mice than standard bone morphometric indices. The high accuracy of the SVML model in classifying SARS-CoV-2 infected mice opens the possibility of translating these biom rkers to the clinical setting for early detection of skeletal changes associated with long-haul COVID. The methods presented here were demonstrated using SARS-CoV-2 as a model system and can also be adapted to other diseases associated with altered bone turnover. Development of machine-learning methods for radiomic applications is a crucial step toward clinically relevant radiomic biomarkers of bone health and provides a non-invasive way of quantifying and monitoring systemic bone loss and evaluating treatment efficacy. [Formula presented]

SARS-CoV-2 Ferritin Nanoparticle Vaccines Elicit Broad SARS Coronavirus Immunogenicity

Background. The zoonotic emergence of SARS-CoV-2 quickly developed into a global pandemic. Multiple vaccine platforms have been advanced to clinical trials and emergency use authorization. The recent emergence of SARS-CoV-2 virus variants with Spike receptor-binding domain (RBD) and N-terminal domain (NTD) mutations, highlights the need for next-generation vaccines that can elicit immune responses that are resilient against Spike mutations. Methods. Using a structure-based vaccine design approach, we developed multiple optimized SARS-CoV-2 nanoparticle immunogens that recapitulate the structural and antigenic profile of the SARS-CoV-2 prefusion spike. We assessed these immunogens in murine immunogenicity studies and in a K18-hACE2 transgenic mouse model with a SARS-CoV-2 challenge. Immune sera from vaccinated mice were assessed for SARS-CoV-2 binding, and neutralization against SARS-CoV-2, variants of concern, and the heterologous SARS-CoV-1 virus. Results. In combination with a liposomal-saponin based adjuvant (ALFQ), these immunogens induced robust binding, ACE2-inhibition, and authentic virus and pseudovirus neutralization. A Spike-Ferritin nanoparticle (SpFN) vaccine elicited neutralizing ID50 titers >10,000 after a single immunization, while RBD-Ferritin (RFN) nanoparticle immunogens elicited ID50 titer values >10,000 values after two immunizations. Purified antibody from SpFN- or RFN-immunized mice was transfused into K18-ACE2 transgenic mice and challenged with a high-dose SARS-CoV-2 virus stock. In order to understand the breadth of vaccine-elicited antibody responses, we analyzed SpFN- and RBD-FN-immunized animal sera against a set of heterologous SARSCoV-2 RBD variants and SARS-CoV RBD. High binding titers with ACE2-blocking activity were observed against SARS-CoV-2 variants and the heterologous SARSCoV-1 RBD. Furthermore, both SpFN- and RFN-immunized animal sera showed SARS-CoV-1 neutralizing ID50 titers of >2000. Conclusion. These observations highlight the importance of SARS-CoV-2 neutralizing antibody levels in providing protection against emerging SARS-like coronaviruses and provide a robust platform for pandemic preparedness. Structure-based design enables development of a SARS-CoV-2 nanoparticle immunogen.

Abstracts from the 21st Australia and New Zealand Microcirculation Society Symposium

The proceedings contain 32 papers. The topics discussed include: characterization of pericyte changes in healthy and type 2 diabetic muscles;using a PDGFR-CREERT2 transgenic mouse line to deplete pericytes in the brain;megadose vitamin C: a new therapeutic to reverse renal microcirculatory dysfunction in sepsis and COVID-19;stroke accentuates age-dependent neutrophil impairment;biological control of adipose tissue repair - implications for healing of surgical wounds;circulating CCR6+ ILC levels are altered in alemtuzumab-treated multiple sclerosis patients;cladribine alters lymphocyte trans-endothelial migration via CD49D expression in multiple sclerosis patients;extracellular vesicles and mimetic technologies for theranostics;and novel phosphorescent stain for microvesicle penetration through brain microvascular endothelium.

An engineered high-affinity ACE2 peptide therapeutically alleviates lung vascular endothelial injury and mortality induced by distinct SARS-CoV-2 variants

Introduction: Vaccine hesitancy and the continuous emergence of SARS-CoV-2 variants of concern that partially escape vaccine-induced immune responses highlight the urgent need for the development of effective therapeutics for COVID-19 patients. Hypothesis: The Engineered high-affinity ACE2 peptide has a therapeutic effect for SARS-CoV-2 and its variant-induced acute lung vascular injury. Methods: K18-hACE2 transgenic mice were inoculated with SARS-CoV-2 WA-1/2020 or P.1 variant of concerns to induce acute lung injury. The sACE22.v2.4-IgG1 was injected once a day for consecutively 7 days starting from 12 hours at 10 mg/kg/day or 24 hours at 15 mg/kg/day postSARS-CoV-2 inoculation. Results: We demonstrated that the higher binding affinity of sACE22.v2.4-IgG1 to S proteins of the original SARS-CoV-2 WA-1/2020 isolate and the B.1.1.7 (Alpha, UK isolate), B.1.351 (Beta, South Africa isolate), P.1 (Gamma, Brazil isolate), and B.1.617.2 (Delta, India isolate) SARS-CoV-2 variants of concern compared to wild-type soluble ACE2. Using humanized K18-hACE2 mice, we found that both SARS-CoV-2 isolate WA-1/2020 and the P.1 variant of concern induced severe acute lung endothelial injury, which resulted in the severe transvascular leak, lung edema, and death. The P.1 variant induced severe lung vascular leak and death at an earlier time point at Day 5 compared to WA-1 at Day 7, consistent with increased viral entry and replication. Treatment with sACE22.v2.4-IgG1 decoy peptide could be initiated as late as 24h after inoculation with a lethal dose of the SARS-CoV-2 WA-1/2020 isolate and profoundly reduces lung vascular injury and mortality For the P.1 variant of concern, ACE22.v2.4-IgG1 decoy peptide markedly improved survival and reduced lung vascular injury if administered early at 12 hours post-inoculation. Conclusion: sACE22.v2.4-IgG1 has an exceptional therapeutic efficacy to SARS-CoV-2 WA-1/2020 and variant P.1 -induced acute lung vascular injury.

Sars-Cov-2 Infection Promotes Endothelial Dysfunction and Thrombosis in a Mouse Model of COVID-19

Coronavirus disease 2019 (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Most infected individuals are asymptomatic or show only mild symptoms, but 20% of infected individuals become severely ill resulting in a 2-5% mortality rate for severe infections. Men, the elderly and patients with comorbidities (such as cardiovascular disease, hypertension, diabetes, and obesity) are more likely to develop severe disease. Clinical features characterizing severe COVID-19 cases include inflammation and thrombosis, but the molecular mechanisms underlying these processes remain elusive. K18 hACE2 transgenic mice express the SARSCoV-2 receptor human angiotensin-converting enzyme 2 (hACE2) under the control of the human cytokeratin 18 (K18) promoter. K18 hACE2 mice express hACE2 in airway epithelial cells and are susceptible to SARS-CoV-1 and SARS-CoV-2 infections. At the dose of 10 5 PFU/mouse, all SARS-CoV-2-infected K18 hACE2 mice rapidly lose weight and succumb to viral infection by 5-6 days post infection. Morbidity and mortality correlated with SARS-CoV-2 replication in the nasal turbinates and lungs. Notably, susceptibility was highly associated with a local and systemic cytokine/chemokine storm. SARS-CoV-2 infection in K18 hACE2 mice recapitulates many of the pathological findings observed in human patients offering a reliable animal model for the study of SARS-CoV-2 pathogenesis. Infection with a lower viral dose (10 4 and 2.5x10 3 PFU/mouse) prolongs the symptomatic phase of the infection, postponing time of death up to 16 days post infection (mortality rate at 10 4 PFU: ~40% in females, 100% in males;mortality rate at 2.5x10 3 PFU: ~30% in females, ~55% in males). At these lower viral doses, K18 hACE2 mouse males exhibited both increased susceptibility to the SARS-CoV-2 infection and more severe disease. Male mice showed increased mortality associated with an increase in weight loss and decrease in body temperature. Disease characteristics showed striking similarities with reported human COVID-19 cases, including severely reduced O 2 saturation. The pathogenesis of severe COVID-19 cases involves both virus-induced cell damage and secondary tissue damage due to a vicious cycle of dysregulated - hyperactive coagulation and inflammatory pathways that present as “a cytokine storm”, endothelial dysfunction, and “immunothrombosis”. Analysis of murine plasma analytes from infected mice revealed additional pathogenetic features resembling SARS-CoV-2 infection in humans. High circulating D-dimer levels are now considered a main predictor of poor outcome of SARS-CoV-2 infection. Notably, we also observed a progressive increase of circulating D-dimer levels in the plasma of K18 hACE2 infected mice peaking at day 7 post infection, suggestive of a hypercoagulable state. Moreover, similar to humans, the increase in soluble thrombomodulin plasma concentration and its correlation with disease severity was indicative of endothelial activation and dysfunction in K18 hACE2 infected mice. SARS-CoV-2 infection-induced changes of coagulation and endothelial activation in mice resulted in a biphasic alteration of endothelial permeability where an initial increase in vascular permeability, peaking at day 5 post infection, was followed by a sudden decrease in Evan's blue dye extravasation in the lung parenchyma and characterized by the appearance of areas of hemorrhagic infarction indicative of thrombotic events. Altogether, our results identify the K18 hACE2 transgenic mouse as an important small animal model to study the molecular mechanisms involved in the derangement of the finely tuned interaction between the immune and coagulation systems associated with severe cases of SARS-CoV-2 infections. Disclosures: Mosnier: Hematherix: Membership on an entity's Board of Directors or advisory committees;Coagulant Therapeutics: Research Funding.

Comparison of SARS-CoV-2 variant lethality in human angiotensin-converting enzyme 2 transgenic mice.

This study compared the lethality of severe acute respiratory syndrome coronavirus 2 variants belonging to the S, V, L, G, GH, and GR clades using K18-human angiotensin-converting enzyme 2 heterozygous mice. To estimate the 50% lethal dose (LD50) of each variant, increasing viral loads (100-104 plaque-forming units [PFU]) were administered intranasally. Mouse weight and survival were monitored for 14 days. The LD50 of the GH and GR clades was significantly lower than that of other clades at 50 PFU. These findings suggest that the GH and GR clades, which are prevalent worldwide, are more virulent than the other clades.

Protective Efficacy of Inactivated Vaccine against SARS-CoV-2 Infection in Mice and Non-Human Primates.

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused more than 96 million infections and over 2 million deaths worldwide so far. However, there is no approved vaccine available for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the disease causative agent. Vaccine is the most effective approach to eradicate a pathogen. The tests of safety and efficacy in animals are pivotal for developing a vaccine and before the vaccine is applied to human populations. Here we evaluated the safety, immunogenicity, and efficacy of an inactivated vaccine based on the whole viral particles in human ACE2 transgenic mouse and in non-human primates. Our data showed that the inactivated vaccine successfully induced SARS-CoV-2-specific neutralizing antibodies in mice and non-human primates, and subsequently provided partial (in low dose) or full (in high dose) protection of challenge in the tested animals. In addition, passive serum transferred from vaccine-immunized mice could also provide full protection from SARS-CoV-2 infection in mice. These results warranted positive outcomes in future clinical trials in humans.

One or two injections of MVA-vectored vaccine shields hACE2 transgenic mice from SARS-CoV-2 upper and lower respiratory tract infection.

Modified vaccinia virus Ankara (MVA) is a replication-restricted smallpox vaccine, and numerous clinical studies of recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases, including COVID-19, are in progress. Here, we characterize rMVAs expressing the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Modifications of full-length S individually or in combination included two proline substitutions, mutations of the furin recognition site, and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) is flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected cells and was recognized by anti-RBD antibody and soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced antibodies, which neutralized a pseudovirus in vitro and, upon passive transfer, protected hACE2 transgenic mice from lethal infection with SARS-CoV-2, as well as S-specific CD3+CD8+IFNγ+ T cells. Antibody boosting occurred following a second rMVA or adjuvanted purified RBD protein. Immunity conferred by a single vaccination of hACE2 mice prevented morbidity and weight loss upon intranasal infection with SARS-CoV-2 3 wk or 7 wk later. One or two rMVA vaccinations also prevented detection of infectious SARS-CoV-2 and subgenomic viral mRNAs in the lungs and greatly reduced induction of cytokine and chemokine mRNAs. A low amount of virus was found in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of low levels of subgenomic mRNAs in turbinates indicated that replication was aborted in immunized animals.

COVID-19 preclinical models: human angiotensin-converting enzyme 2 transgenic mice.

Coronavirus disease 2019 (COVID-19) is a declared pandemic that is spreading all over the world at a dreadfully fast rate. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the pathogen of COVID-19, infects the human body using angiotensin-converting enzyme 2 (ACE2) as a receptor identical to the severe acute respiratory syndrome (SARS) pandemic that occurred in 2002-2003. SARS-CoV-2 has a higher binding affinity to human ACE2 than to that of other species. Animal models that mimic the human disease are highly essential to develop therapeutics and vaccines against COVID-19. Here, we review transgenic mice that express human ACE2 in the airway and other epithelia and have shown to develop a rapidly lethal infection after intranasal inoculation with SARS-CoV, the pathogen of SARS. This literature review aims to present the importance of utilizing the human ACE2 transgenic mouse model to better understand the pathogenesis of COVID-19 and develop both therapeutics and vaccines.