Perinatal SARS-CoV-2 infection and vaccination: new insight

Cumulative data regardingCOVID-19 infection during pregnancy have demonstrated the ability of SARS-CoV-2 to infect the placenta. However, the mechanisms of SARS-CoV-2 placental viral entry are yet to be defined. SARS-CoV-2 infects cells by binding to the ACE2 receptor. However, SARS-CoV-2 cell entry also requires co-localization of spike protein cleavage by the serine protease TMPRSS2. However, the co-expression of ACE2 and TMPRSS2 in placental cells is debated, raising the question of whether potential non-canonical molecular mechanismsmay be involved in SARS-CoV-2 placental cells' viral entry. Although published data regarding the ability of the SARS-CoV- 2 to infect the fetus are contradicting, the placenta appears to be an immunological barrier to active SARS-CoV-2 infection and vertical transmission;however, the mechanism is unclear. Our experiments demonstrated the ability of the SARS-CoV-2 virus to directly infect the placenta and induce transcriptomic responses in COVID-positive mothers. These transcriptomic responses were characterized by differential expression of specific mRNAs and miRNAs associated with SARS-CoV-2 infection, with induction of specific placental miRNAs that can inhibit viral replication. Failure in such mechanisms may be associated with vertical transmission. Since the start of the COVID-19 pandemic, the COVID-19 mRNA vaccines have been widely used to reduce the morbidity and mortality of SARS-CoV-2 infection. Historically, non-live vaccines have not caused any harm to pregnant mothers;however, it is unclear whether our current understanding of the effects of non-live vaccines serves as a reliable precedent owing to the novel technology used to create these mRNA vaccines. Since there are no definitive data on the possible biodistribution of mRNA vaccines to the placenta, the likelihood of vaccine mRNA reaching the fetus remains uncertain. Little has been reported on the tissue localization of the lipid nanoparticles (LNPs) after intramuscular (IM) administration of the mRNA vaccine. The biodistribution of LNPs containing the mRNA vaccine has been investigated in animal models but not humans. In the murine model, the vaccine LNPs were rapidly disseminated to several organs, including the heart, liver, kidney, lung, and spleen, following IM administration. However, no traditional pharmacokinetic or biodistribution studies have been performed with the mRNA vaccines, including possible biodistribution to breast milk or the placenta.

Cyclic [68Ga] and [18F]AlF-labeled peptides for specific detection of human angiotensin-converting enzyme 2

Objectives: In this study, we developed angiotensin-converting enzyme 2 (ACE2)-specific, peptide-derived 68Ga- and 18F-labeled radiotracers, motivated by the hypotheses that ACE2 is an important determinant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) susceptibility and that modulation of ACE2 in coronavirus disease 2019 (COVID-19) drives severe organ injury. Our current efforts are focusing on broader dissemination of ACE2-targeted PET radiotracers based on chelation of [18F]AlF enabling advanced murine and potentially human studies. Method(s): A series of NOTA-conjugated peptides derived from the known ACE2 inhibitor DX600 were synthesized, with variable linker identity. Since DX600 bears 2 cystine residues, both linear and cyclic peptides were studied. An ACE2 inhibition assay was used to identify lead compounds, which were labeled with 68Ga and 18F-AlF to generate the corresponding peptide radiotracers (68Ga-NOTA-PEP). The most potent 68Ga and 18F-AlF DX600 derivatives were subsequently studied in a humanized ACE2 (hACE2) transgenic model. Result(s): Cyclic DX-600-derived peptides had markedly lower half-maximal inhibitory concentrations than their linear counterparts. The 3 cyclic peptides with triglycine, aminocaproate, and polyethylene glycol linkers had calculated half-maximal inhibitory concentrations similar to or lower than the parent DX600 molecule. Peptides were readily labeled with 68Ga and 18F-AlF, and the biodistribution of both tracers was determined in an hACE2 transgenic murine cohort. Pharmacologic concentrations of coadministered NOTA-PEP (blocking) showed a significant reduction of 68Ga-NOTA-PEP4 signals in the heart, liver, lungs, and small intestine. Ex vivo hACE2 activity in these organs was confirmed as a correlate to in vivo results. The biodistribution of both tracers was similar, with apparent blocking observed in the lungs using the 18F-AlF peptide that needs to be verified via additional experiments. Conclusion(s): NOTA-conjugated cyclic peptides derived from the known ACE2 inhibitor DX600 retain their activity when N-conjugated for 68Ga or 18F-AlF chelation. In vivo studies in a transgenic hACE2 murine model using the lead tracer, 68Ga-NOTA-PEP4, showed specific binding in the heart, liver, lungs and intestine-organs known to be affected in SARS-CoV-2 infection. Blocking studies using the 18F-AlF labeled correlate showed modulation of PET signals in the normal lungs. These results suggest that 68Ga-NOTA-PEP4 or the 18F-AlF correlate could be used to detect organ-specific suppression of ACE2 in SARS-CoV-2-infected murine models and COVID-19 patients.Copyright © 2023 Southern Society for Clinical Investigation.

In vivo murine studies demonstrate that neutrophil activation by anti-NAP2 antibodies contributes to vaccine-induced immune thrombocytopenia and thrombosis (VITT)

Background: VITT involves thrombocytopenia and thrombosis post-initial anti-SARS-CoV-2 adenoviral vaccination. Most patients are found to have platelet-activating antibodies to the chemokine, platelet factor 4 (PF4) in the absence of heparin. VITT antibodies differ from those in heparin-induced thrombocytopenia (HIT), in which PF4 is bound to heparin. Distinct epitope sites on PF4 for VITT and HIT antibodies were defined (PMID34233346). We noted that the VITT antigenic site is conserved in mouse (m) PF4, and in the platelet-specific chemokine neutrophil-activating peptide 2 (NAP2), both human and mouse. We observed that VITT antibodies bind strongly to NAP2. In an active patient with VITT, we found that VITT antibodies circulate as immune complexes containing either PF4 or NAP2. Importantly, VITT antibodies plus NAP2 activates platelets. Aim(s): We tested in a passive-immunization murine model with VITT antibodies the ability to induce neutrophil-endothelial activation as an indicator of a prothrombotic state and identify the chemokines involved. Method(s): We studied two systems: A femoral vein and a cremaster venule model, using confocal intravital imaging and labeled neutrophils. VITT antibodies were infused into mice transgenic for FcgammaRIIA and lacking PF4 (FcgammaRIIA+/mPF4-/-). Result(s): This led to an immediately reduced neutrophil rolling by ~80% (14-3 m/sec) (Figure 1A,B). Subsequent infusion of PF4 slowed neutrophil rolling by another ~80% (3-0.6 m/sec). In contrast, VITT antibodies did not slow neutrophil speed in FcgammaRIIA+/ mPF4-/-/ mNAP2-/-mice (Figure 1C). Conclusion(s): These data suggest that both NAP2 and PF4 contribute to thrombosis in VITT and may explain the pathogenesis of VITT in patients with no detectable anti-PF4 antibodies. VITT may be prothrombotic because it involves co-activation of neutrophils via NAP2 by way of CXCR2 and FcgammaRIIA. Targeting NAP2 pathobiology may enhance understanding of the pathogenesis of VITT and lead to new therapeutics.

Potential Original Drug for Aspergillosis: In Vitro and In Vivo Effects of 1-N,N-Dimethylamino-5-Isocyanonaphthalene (DIMICAN) on Aspergillus fumigatus.

As the recent outbreak of coronavirus disease 2019 (COVID-19) has shown, viral infections are prone to secondary complications like invasive aspergillosis with a high mortality rate, and therefore the development of novel, effective antifungals is of paramount importance. We have previously demonstrated that 1-amino-5-isocyanonaphthalene (ICAN) derivatives are promising original drug candidates against Candida strains (Patent pending), even against fluconazole resistant C. albicans. Consequently, in this study ICANs were tested on Aspergillus fumigatus, an opportunistic pathogen, which is the leading cause of invasive and systematic pulmonary aspergillosis in immunosuppressed, transplanted and cancer- or COVID-19 treated patients. We have tested several N-alkylated ICANs, a well as 1,5-naphthalene-diisocyanide (DIN) with the microdilution method against Aspergillus fumigatus strains. The results revealed that the diisocyanide (DIN) was the most effective with a minimum inhibitory concentration (MIC) value as low as 0.6 µg mL-1 (3.4 µM); however, its practical applicability is limited by its poor water solubility, which needs to be overcome by proper formulation. The other alkylated derivatives also have in vitro and in vivo anti-Aspergillus fumigatus effects. For animal experiments the second most effective derivative 1-N, N-dimethylamino-5-isocyanonaphthalene (DIMICAN, MIC: 7-8 µg mL-1, 36-41 µM) was selected, toxicity tests were made with mice, and then the antifungal effect of DIMICAN was tested in a neutropenic aspergillosis murine model. Compared to amphotericin B (AMB), a well-known antifungal, the antifungal effect of DIMICAN in vivo turned out to be much better (40% vs. 90% survival after eight days), indicating its potential as a clinical drug candidate.

Intranasal administration of a VLP-based vaccine induces neutralizing antibodies against SARS-CoV-2 and variants of concern

Background: The highly contagious SARS-CoV-2 is mainly transmitted by respiratory droplets and aerosols. Consequently, people are required to wear masks and maintain a social distance to avoid spreading of the virus. Despite the success of the commercially available vaccines, the virus is still uncontained globally. Given the tropism of SARS-CoV-2, a mucosal immune reaction would help to reduce viral shedding and transmission locally. Only seven out of hundreds of ongoing clinical trials are testing the intranasal delivery of a vaccine against COVID-19. Methods: In the current study, we evaluated the immunogenicity of a traditional vaccine platform based on virus-like particles (VLPs) displaying RBD of SARS-CoV-2 for intranasal administration in a murine model. The candidate vaccine platform, CuMVTT -RBD, has been optimized to incorporate a universal T helper cell epitope derived from tetanus-toxin and is self-adjuvanted with TLR7/8 ligands. Results: CuMVTT-RBD vaccine elicited a strong systemic RBD- and spike- IgG and IgA antibodies of high avidity. Local immune response was assessed and our results demonstrate a strong mucosal antibody and plasma cell production in lung tissue. Furthermore, the induced systemic antibodies could efficiently recognize and neutralize different variants of concern (VOCs) of mutated SARS-CoV-2 RBDs. Conclusion: Our data demonstrate that intranasal administration of CuMVTT-RBD induces a protective systemic and local specific antibody response against SARS-CoV-2 and its VOCs.

Nanoparticles: Efficient, safe and affordable platforms for developing vaccines against coronaviruses

Introduction: Vaccines need to be rationally designed to be delivered to the immune system for maximizing induction of dynamic immune responses. Virus-like nanoparticles (VLPs) are ideal platforms for such 3D vaccines. Coronaviruses have recently gained a lot of attention, due to the ongoing pandemic caused by SARS-CoV-2 and previous endemics by MERS-CoV. Methods: We have provided proof of concepts in murine models for effective development of VLP-based vaccines against MERS-CoV and SASR-CoV-2. We have used chemical conjugation or genetic fusion techniques to display receptor-binding domain or motif on our immunologically optimized (CuMVTT -VLPs). These VLPs incorporate a tetanus toxin epitope and ssRNA, TLR7/8 ligands. The vaccines were tested in murine models. Results: The vaccines are stable for more than a year at 4°C and highly scalable. Vaccination using subcutaneous or intranasal routes are feasible with nanoparticles. We demonstrated that these vaccines are highly immunogenic in mice as well as rabbits and can induce high avidity antibodies compared to convalescent human sera. Furthermore, the induced antibodies are cross-reactive with different VoCs (in case of SARS-CoV-2). The longevity of the induced immune response lasted longer than 120 days. Conclusion: Collectively, we show that VLP-based vaccines can efficiently induce high specific anti-RBD and spike antibodies that effectively neutralize different Coronaviruses and their VoCs. As Coronaviruses represent a continuous global threat to human health, it seems rational to further develop these vaccines.

PET imaging of innate immunity activation In Vivo with redox-tuned 4-[ 18F]fluoro-1-naphthol

High redox potential reactive oxygen and nitrogen species (ROS/RNS), such as O2 free radicals, superoxide, and hypochlorous acid, generated by activities of the NADPH oxidase-2 (NOX2)/myeloperoxidase (MPO) axis and related enzymes, are key effector molecules of innate immunity in physiological and diseased inflammatory states. Other lower energy species (H2O2, NO) provide adjuvant signaling functions. NOX2- and MPO-derived high energy radicals are known to oxidize naphthol species, wherein the naphthol products bind to proximate proteins and activated myeloid cells. Herein, we present 4-[18F]fluoro-1-naphthol ([18F]4FN), a novel redox-tuned radiopharmaceutical that selectively detects by positron emission tomography (PET) high energy radicals produced by activated innate immunity. The products of human MPO plus H2O2 , but not H2O2 alone, rapidly and completely oxidized [18F]4FN. All-trans-retinoic acid-differentiated HL-60 'neutrophil-like' human cells activated with phorbol-12-myristate-13-acetate (PMA) retained [ 18F]4FN 5-fold over unstimulated cells. 4-ABAH, an MPO-specific inhibitor, or DPI, a broad oxidase inhibitor, blocked cellular retention by >95%. [18F]4FN PET/CT imaging readily discriminated foci of inflammation in vivo in three distinct murine models of acute inflammation: endotoxin-induced whole-body toxic shock, PMA-induced mild contact dermatitis of the ear, and lipopolysaccharide (LPS)-induced ankle arthritis. Mechanistically, in mice in vivo, 4-ABAH reduced inflammationinduced [18F]4FN retention, and Cybb-/- (Nox2-/-) gene-deletion strongly and significantly abrogated PMA-induced [18F]4FN retention. Thus, [18F]4FN shows promise as a robust redox-tuned reporter for imaging activation states of innate immunity by PET/CT, is ready for translation. [18F]4FN PET imaging may find application in a variety of inflammatory states associated with cancer therapy, immunotherapy-related adverse events, as well as other diseases, including arthritis, hepatitis, atherosclerosis, COVID-19, as well as up-staging and monitoring multi-organ inflammation.

STRUCTURAL CHARACTERIZATION of DNA-ENCODED HIV VACCINES INDUCED NEUTRALIZING ANTIBODY

Background: Rapid and large-scale deployment of COVID-19 mRNA vaccines highlights the potential utility of developing nucleic acid vaccines (such as RNA and DNA vaccines) against infectious diseases, including HIV-1. However, as compared to SARS-CoV-2, HIV-1 pose some unique challenges-induction of neutralizing antibodies (NAbs) against HIV-1 (frequently a correlate of protection) requires presentation of trimeric and highly conformational epitopes to the immune system, and whether nucleic acid vaccines can enable direct in vivo production of antigens that retain critical antigenic profile has not yet been elucidated. Additionally, it was previously reported that Tier 2 NAbs cannot be induced in mice due to a lack of antibody repertoire, and vaccine studies were suggested to be performed in larger mammals such as rabbits/NHPs, inadvertently slowing down and increasing the costs of preclinical HIV-1 vaccine studies. Methods: In our study, we used the Antigen Conformation Tracing In Vivo by ELISA (ACTIVE) assay developed in house to characterize antigenic profiles of vaccines produced in vivo (from transfected muscle tissues). We analyzed induced cellular responses, using stimulation with overlapping peptides followed by intracellular cytokine staining and IFN-g ELIspot assays. We analyzed induced humoral responses by using both binding ELISA assays and TZM-BL based neutralizing assays, and attempted to map induced NAb epitopes by engineering selectively mutated pseudovirus. We performed antigen-specific B-cell sorting, and used the 10x genomics pipeline to characterize antibody sequences of proliferating B-cell clones. Results: We confirmed that in vivo produced vaccines retained key trimeric conformational epitopes and glycan profiles. Compared to protein vaccination, DNA vaccination uniquely and strongly induced both TFH, CD4+, CD8+ T-cell responses, and Tier 2 NAbs mapped to a previously unreported Env C3/V5 epitope. 5 unique NAbs were isolated, and confirmed to bind to the epitope using a Cryo-EM structure of NAb-MD39 complex at 3.8Å resolution. Conclusion: Our study confirmed that with appropriate vaccine delivery technology, murine models can be appropriately used for HIV-1 vaccine studies aimed at generating NAb responses. In addition, beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines.

INTERIM ANALYSIS of A PHASE i STUDY of PRTX007: SAFETY, PK, and PD RESPONSE

Background: PRTX007 is a prodrug of PRX034, a novel TLR7 agonist, for treatment of respiratory viral diseases including SARS-CoV-2. PRX034 activates plasmacytoid dendritic cells to preferentially synthesize poly sub-type interferons while minimizing NFκΒ-mediated proinflammatory factors. Pre-clinical studies have demonstrated decoupling of these two normally linked processes in human PBMCs in vitro and in cynomolgus monkeys in vivo. Antiviral activity is preserved, as demonstrated by inhibition of cytopathicity and viral replication in RNA viruses by conditioned media from PRX034-treated PBMCs. Efficacy and safety have been demonstrated in a murine model of RSV infection. An interim analysis of the first-in-human study in healthy volunteers is presented here. Methods: Phase I, single-center, prospective, randomized, double-blind study of 8 single-ascending dose (SAD) cohorts and 4 multiple-ascending dose (MAD) cohorts of PRTX007 administered orally to healthy adult subjects. Primary objective was safety and tolerability. Secondary objectives were to assess pharmacokinetics (PK) and pharmacodynamics (PD). This interim analysis focuses on the SAD portion of the study over 8 dose levels from 50-600mg. Safety through all SAD cohorts, PK up to the 500 mg cohort, PD up to the 400 mg cohort are presented. The MAD cohort safety data from 3 of 4 planned cohorts are also presented. Results: Treatment-related adverse events (AEs) include mild to moderate headache. Severity or frequency is not dose related, is of short duration and is not associated with systemic symptoms. One participant in the 400 mg cohort had asymptomatic mild sinus tachycardia, attributed to PRTX007. Two subjects in the second MAD cohort at 300 mg had mild increases in ALT that rapidly resolved after treatment. PK analysis demonstrated efficient conversion of PRTX007 to PRX034 with systemic exposure to drug and prodrug increasing proportionally with dose. Duration of systemic exposure to PRX034 at pharmacologically active levels is consistent with activation of innate immune response without counter-regulation (Panel A). Exposure-dependent PD response measured by induction of interferon stimulated genes in the blood is observed at drug AUC > 4,300 hr∗ng/ml (Panel B);duration of induction >24 hours (Panel C). Conclusion: Interim analysis of PRTX007 demonstrates a favorable safety profile with dose-dependent systemic exposure and demonstrated activation of innate immune response.

Effective Interferon Lambda Treatment Regimen To Control Lethal MERS-CoV Infection in Mice.

Effective broad-spectrum antivirals are critical to prevent and control emerging human coronavirus (hCoV) infections. Despite considerable progress made toward identifying and evaluating several synthetic broad-spectrum antivirals against hCoV infections, a narrow therapeutic window has limited their success. Enhancing the endogenous interferon (IFN) and IFN-stimulated gene (ISG) response is another antiviral strategy that has been known for decades. However, the side effects of pegylated type-I IFNs (IFN-Is) and the proinflammatory response detected after delayed IFN-I therapy have discouraged their clinical use. In contrast to IFN-Is, IFN-λ, a dominant IFN at the epithelial surface, has been shown to be less proinflammatory. Consequently, we evaluated the prophylactic and therapeutic efficacy of IFN-λ in hCoV-infected airway epithelial cells and mice. Human primary airway epithelial cells treated with a single dose of IFN-I (IFN-α) and IFN-λ showed similar ISG expression, whereas cells treated with two doses of IFN-λ expressed elevated levels of ISG compared to that of IFN-α-treated cells. Similarly, mice treated with two doses of IFN-λ were better protected than mice that received a single dose, and a combination of prophylactic and delayed therapeutic regimens completely protected mice from a lethal Middle East respiratory syndrome CoV (MERS-CoV) infection. A two-dose IFN-λ regimen significantly reduced lung viral titers and inflammatory cytokine levels with marked improvement in lung inflammation. Collectively, we identified an effective regimen for IFN-λ use and demonstrated the protective efficacy of IFN-λ in MERS-CoV-infected mice. IMPORTANCE Effective antiviral agents are urgently required to prevent and treat individuals infected with SARS-CoV-2 and other emerging viral infections. The COVID-19 pandemic has catapulted our efforts to identify, develop, and evaluate several antiviral agents. However, a narrow therapeutic window has limited the protective efficacy of several broad-spectrum and CoV-specific antivirals. IFN-λ is an antiviral agent of interest due to its ability to induce a robust endogenous antiviral state and low levels of inflammation. Here, we evaluated the protective efficacy and effective treatment regimen of IFN-λ in mice infected with a lethal dose of MERS-CoV. We show that while prophylactic and early therapeutic IFN-λ administration is protective, delayed treatment is detrimental. Notably, a combination of prophylactic and delayed therapeutic administration of IFN-λ protected mice from severe MERS. Our results highlight the prophylactic and therapeutic use of IFN-λ against lethal hCoV and likely other viral lung infections.

Bacterial components suppress experimental acute lung injury through regulatory T-cells

Introduction: The induction of regulatory T cells (Tregs) is indicated as a potential therapeutic strategy in inflammatory lung diseases including, asthma, viral-induced pneumonia, viral-induced acute lung injury (ALI), severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and SARSCoV- 2-induced ALI. We previously identified that components of the bacteria Streptococcus pneumoniae (T + P) are able to increase Tregs to suppress experimental allergic airways disease, however, this mechanism of suppression and therapy has not been examined in ALI. Methods: We established a murine model of ALI using aerosolized LPS (100 μg/ml) in BALB/c mice. ALI was measured by the presence of neutrophils in the airways up to 96 hours post-exposure, and Tregs and dendritic cells were assessed by flow cytometry. To assess the therapeutic of T + P in ALI and the mechanisms involved, the combination was administered prior to LPS exposure in the absence or presence of anti-CD25. Results: Treatment with T + P significantly reduced total airway inflammation and suppressed the neutrophil chemokine C-X-C motif chemokine ligand 1 (Cxcl1) compared to Saline+LPS alone in experimental ALI. The numbers of Tregs were reduced in experimental ALI model and were restored by T + P treatment. Depletion of Tregs with anti- CD25 confirmed that the suppressive effects of T + P on ALI was through the induction of Tregs. Conclusion: Treatment with S. pneumoniae components T + P suppresses neutrophilic inflammation in ALI through immunoregulatory mechanisms that involve Tregs and may be a novel treatment for ALI including in COVID-19.

Design and Immunogenicity of a Pan-SARS-CoV-2 Synthetic DNA Vaccine

Background. First-generation COVID-19 vaccines are matched to spike protein of the Wuhan-H1 (WT) strain. Convalescent and vaccinee samples show reduced neutralization of SARS-CoV-2 variants of concern (VOC). Next generation DNA vaccines could be matched to single variants or synthetically designed for broader coverage of multiple VOCs. Methods. The synthetic consensus (SynCon®) sequence for INO-4802 SARSCoV-2 spike with focused RBD changes and dual proline mutations was codon-optimized (Figure 1). Sequences for wild-type (pWT) and B.1.351 (pB.1.351) were similarly optimized. Immunogenicity was evaluated in BALB/c mice. Pre-clinical efficacy was assessed in the Syrian Hamster model. Figure 1. Design Strategy for INO-4802 Results. INO-4802 induced potent neutralizing antibody responses against WT, B.1.1.7, P.1, and B.1.351 VOC in a murine model. pWT vaccinated animals showed a 3-fold reduction in mean neutralizing ID50 for the B.1.351 pseudotyped virus. INO-4802 immunized animals had significantly higher (p = 0.0408) neutralizing capacity (mean ID50 816.16). ID50 of pB.1.351 serum was reduced 7-fold for B.1.1.7 and significantly lower (p = 0.0068) than INO-4802 (317.44). INO-4802 neutralized WT (548.28) comparable to pWT. INO-4802 also neutralized P.1 (1026.6) (Figure 2). pWT, pB.1.351 or INO-4802 induced similar T-cell responses against all variants. INO-4802 skewed towards a TH1-response. All hamsters vaccinated with INO-4802 or pB.1.351 were protected from weight loss after B.1.351 live virus challenge. 4/6 pWT immunized hamsters were completely protected. pWT immunized hamsters neutralized WT (1090) but not B.1.351 (39.16). INO-4802 neutralized both WT (672.2) and B.1.351 (1121) (Figure 3). We observed higher increase of binding titers following heterologous boost with INO-4802 (3.6 - 4.4 log2-fold change) than homologous boost with pWT (2.0 - 2.4 log2 fold change) (Figure 4). Conclusion. Vaccines matching single VOCs, like pB.1.351 and pWT, elicit responses against the matched antigen but have reduced cross-reactivity. Presenting a pan-SARS-CoV-2 approach, INO-4802 may offer substantial advantages in terms of cross-strain protection, reduced susceptibility to escape mutants and non-restricted geographical use.

LUNG ACE2 AND TMPRSS2 GENE EXPRESSION VARIES WITH ALLERGEN EXPOSURES IN MURINE MODELS OF ASTHMA

Introduction: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires angiotensin-converting enzyme 2 (ACE2) and the transmembrane protease, serine 2 (TMPRSS2) for cell entry. Prior studies have reported that allergen exposure can downregulate ACE2 expression. Here, we sought to determine if exposure to distinct combinations of allergens results in the differential expression of ACE2 and TMPRSS2 in the mouse lung. Methods: We utilized three established murine models of asthma: 1. Alum/Ovalbumin (OVA) model;2. House dust mite (HDM)/OVA model;3. Mixed-allergen (MA) model using OVA, HDM, Aspergillus fumigatus and Alternaria alternata. Phosphate-buffered saline (PBS) treated mice were used as controls. Lung RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol, and complementary DNA was synthesized. Quantitative PCR (qPCR) was performed 24 hours after the last challenge utilizing validated primers for ACE2 and TMPRSS2. Analysis was performed using one-way ANOVA. Results: Here we report that ACE2 mRNA expression was lower in the MA and Alum/OVA-treated mice compared to the controls (p-value < 0.0001). No difference was seen in the ACE2 mRNA expression between HDM/OVA and PBS-treated mice. Furthermore, HDM/OVA-treated mice expressed higher levels of TMPRSS2 mRNA compared to controls (p-value < 0.01). No difference was seen in the TMPRSS2 mRNA expression between the MA or Alum/OVA, and the PBS-treated mice. Conclusion: The exposure to distinct combinations of allergens results in unique patterns of ACE2 and TMPRSS2 gene expression in the mouse lung. Further studies are required to evaluate the effects of allergen exposure with the susceptibility to SARS-CoV-2 infection.

Intraocular injection of KH902 alleviates retinal hypoxia in a mouse model of oxygen-induced retinopathy

Inhibition of vascular endothelial growth factor (VEGF) has been widely applied in anti-neovascularization therapies. As a novel anti-VEGF agent, KH902 (conbercept) is designed to restrain pathological angiogenesis. However, the effects of KH902 on retinal hypoxia have not been well studied. In a mouse model of oxygen-induced retinopathy (OIR), we assessed retinal hypoxia at postnatal days 14 (P14) and P17, as well as retinal neovascularization (RNV) at P17. In addition, we evaluated the protein level of VEGF and galectin-1 (Gal-1). Changes of the neuroretinal structure were also examined. Our results indicated that KH902 could remit retinal hypoxia in OIR at P14 and P17, which was an exciting novel finding for KH902 function. Additionally, we confirmed that KH902 markedly reduces RNV. Our results indicated that administration of KH902 downregulated VEGF expression, as well as Gal-1. Damage of neuroretinal structure after KH902 injection was not observed, which was also an encouraging result. Our study suggests that KH902 plays a role in alleviating retinal hypoxia and that it could be used for the treatment of other ncovascular ocular diseases.

Platelets factor 4 (PF4) binding enhances in vitro neutrophil extracellular traps (NET) capture of SARs-CoV- 2: Therapeutic implications

Background: SARs-CoV- 2 infection recruits high numbers of neutrophils that extrude neutrophil extracellular traps (NETs), webs of extracellular DNA coated with citrullinated histones (cit-His) and antimicrobial proteins. NETs have also been shown to entrap virions, concentrate antiviral proteins, and inactivate viruses. However, when NETs are degraded, they release NET degradation products (NDPs) such as cit-His, cell-free (cf) DNA, myeloperoxidase (MPO) and neutrophil elastase (NE) that can be toxic to the host. Our group and others have found that NETs and NDPs are highly prominent in patients with severe COVID-19 and are associated with the development of respiratory failure (Figure 1). Platelet factor 4 (PF4) is a highly-positively charged, platelet-specific chemokine that aggregates polyanionic molecules like heparin and DNA. We have shown that PF4 binds to NETs, reducing the release of NDPs by preventing NET digestion by circulating nucleases. Importantly, PF4-NET complexes markedly enhance gram-positive and -negative bacterial entrapment, likely by bridging the negatively charged polyanionic phosphoribose backbone of the NET DNA scaffold to polyanionic surface molecules in the bacterial cell wall. Treatment with PF4 improved outcomes in lipopolysaccharide endotoxemia and cecal ligation and puncture models of murine sepsis. Objectives: The objective of this study was to investigate whether PF4 binding to NETs is similarly protective in SARs-CoV- 2 infection by preventing the degradation of NETs and by enhancing NET-mediated viral capture. Methods: We generated NET-lined microfluidic channels. Neutrophils were isolated from healthy human donors, adhered to fibronectin-coated channels, and incubated with phorbol myristate acetate (PMA) to induce the release of NETs. Channels were then treated with buffer alone or PF4 (100 μg/ml) to compact NETs, after which gamma-irradiated SARS-CoV- 2 (1 x 107 PFU) were infused at 2 dynes/cm2 for 1 hour. Viral particles were then labeled with SARS-CoV- 2 guinea pig antiserum and visualized with a fluorescently-labeled secondary antibody. Viral binding to NETs was quantified using confocal microscopy. Results: Similar to that seen with bacterial attachment to NETs, we observed scant viral binding to non-compact NETs. In contrast, there was abundant binding of SARs-CoV- 2 aggregates to PF4 compacted NETs (Figure 2). Conclusions: These findings demonstrate that PF4 plays a crucial role in NET-mediated viral capture and suggest that PF4-NET complexes may be part of the physiologic mechanism by which viral spread is contained in the host. Moreover, we have previously shown that an Fc-modified version of KKO, a monoclonal antibody directed against complexes of PF4 and polyanions, markedly enhanced the protective effects of PF4 in vitro and in murine models of sepsis. Therefore, we will examine whether PF4 plus modified KKO infusions are able to limit SARS-CoV- 2 viremia, preventing the pneumonitis and multi-system organ dysfunction of severe COVID-19. (Figure Presented).

Elevated Central Carbon Metabolism - a Hallmark for Senescent Cells in Aging Human Hematopoietic Stem Cell Compartment

Comprehensive proteomic studies of HSC derived from bone marrow of healthy human subjects (n = 59) in different age groups (range: 20 - 72 years) showed that aging HSCs are characterized not only by myeloid lineage skewing, senescence associated secretory phenotype (SASP), accumulation of reactive oxygen species (ROS), anti-apoptosis, but prominently by elevated glycolysis, glucose uptake, and accumulation of glycogen. This is caused by a subset of HSC that has become more glycolytic than others and not on a per cell basis. Subsequent comparative transcriptome studies of HSCs from human subjects >60 years versus those from <30 years have confirmed this association of elevated glycolysis with aging transcriptome signature. Provided with this background and based on glucose metabolism levels, we have developed a method to isolate human HSCs (CD34+ cells) from bone marrow into three distinct subsets with high, intermediate, and low glucose uptake (GU) capacity (GU high, GU inter, GU low). For human subjects >60 years old (n=9), the proportions of these subsets are: GU high= 5.4+3.5 %, GU inter= 66.4+22.5 %, GU low= 28.2+21.7 %. For subjects <30 years (n=5), the proportions are GU high= 1.7+1.5 %, GU inter= 66.5+36.9 %, GU low= 31.8+36.7. Single-cell RNA-sequencing (scRNA-seq) studies and gene ontology analysis of biological processes revealed that, compared to the GU inter and GU low subsets, the GU high cells showed a significantly higher expression of genes involved in myeloid development, inflammation response (AIF1, CASP2, ANXA1, ZFP36), anti-apoptosis (GSTP1, NME1, BCL2, DMNT1, BAX), cell cycle checkpoint (MCL1, CDK1, CDK4, EIF5A), histone regulation (BCL6, EGR1, KDM1A, MLLT3), b-galactosidase, and significantly lower expressions of genes involved in lymphoid development, and of MDM4, MDM2, FOXP1, SOX4, RB1. Functional studies indicated that the glycolytic enzymes were elevated in elderly HSCs, and the GU low subset corresponded to primitive and more pluripotent HSCs than the GU interand GU high subsets. Pathway analyses have then demonstrated that the GU high subset is associated with up-regulated p53 as well as JAK/STAT signaling pathways, characteristic of senescent HSCs observed in murine models. Applying Gene Set Enrichment Analysis (GSEA) algorithms, we have compared the scRNA-seq data of CD34+ cells derived from young (<30 years) versus older (>60 years) subjects, as well as the scRNA-seq data from GU high subset versus GU inter and GU lowsubsets from each individual subject (n = 6). The results are shown in Figure 1. In analogy to the comparison between old (>60 years) versus young (<30 years) HSCs (CD34+ cells), GSEA of the GU high versus GU inter and GU low subsets shows the same pattern of changes - significant upregulation of gene-set expressions for (a) inflammatory response (b) G2M checkpoint, (c) MTORC1, (d) ROS, (Fig. 1B), (e) allograft rejection;and down-regulation of gene-set expressions for (f) pluripotency, (g) androgen response, (h) UV response (Fig. 1C) as well as (i) interferon-a induction during SARS-CoV2-infection (data not shown in Fig. 1). Thus, our novel findings of elevated glycolysis coupled with significant activation of MTORC1 in the senescent cells of the HSC compartment have provided evidence for the important role of calorie restriction (CR) for healthy aging of HSCs. In numerous animal models, aging has been shown to be driven by the nutrient-sensing MTORC1 network. In animal models of aging, CR has been reported to deactivate the MTOR pathway, thus slowing aging and delaying diseases of aging. Conclusion: In a series of multi-omics studies, we have demonstrated that the GU high subset is identical to the senescent cells (SCs) in human HSC compartment. Studies in animal models have shown that SCs in murine bone marrow are responsible for driving the aging process, and elimination of this subset by inhibitors of anti-apoptotic factors is able to rejuvenate hematopoiesis in mice. Our present results have provided cellular and molecular evidence that SCs in human HSC compartment re also dependent on anti-apoptotic factors, elevated MTORC1 as well as increased glycolysis for survival. Inhibition of MTORC1 or glycolysis, either by specific inhibitors or by CR, may eliminate senescent HSCs and promote rejuvenation of human hematopoiesis. [Formula presented] Disclosures: No relevant conflicts of interest to declare.

Very-High Dose Dexamethasone Mobilizes Endogenous Bi-Specific Gamma Delta+ NKT Cells

Dexamethasone has been widely used since its initial approval by the FDA in 1958, either individually or as part of a therapeutic regimen for a variety of diseases and disorders, including lymphoma and leukemia and most recently, COVID-19 mediated disease. During a preclinical experiment with A20 B-cell lymphoma bearing mice, a suprapharmacologic dose of dexamethasone phosphate, equivalent to a Human (Equivalent) Dose of 17.5 mg/kg, was inadvertently administered. Blood samples were collected and analyzed by flow cytometry, revealing the presence of a new cell 48 hours after dosing. Subsequent experiments confirmed this finding following a single dose of AVM0703. This cell has since been identified as a bi-specific gamma-delta+ NKT cell, or AVM-NKT cell. One of the challenges of being able to deliver suprapharmacologic dexamethasone doses was the drug product itself. These limitations led to the development of a new drug product, AVM0703, which permits the safe administration of the doses necessary to mobilize these cells. AVM0703 is supplied as a sterile, single-use 50 mL, 24 mg/mL solution for infusion, without preservatives. The ability to rapidly mobilize and activate these cells following a single dose of AVM0703 in as little as 6 hours is the subject of an on-going clinical trial, in patients with lymphoid malignancies (NCT04329728), specifically no-option, R/R ALL, MCL, DLBCL, Primary Mediastinal Large B-cell, Burkitt, CLL/SLL and B-or T-ALL. The study consists of 2-parts, dose-escalation to determine the Phase 2 dose, followed by an adaptive-design, expansion cohort study in the same patient population. Concurrently, clinical data has also been obtained from Expanded Access-Single Patient INDs. Based on the murine model, a theoretically effective HED was determined to be at least 18 mg/kg. Because the maximum dose approved for generic injectable dexamethasone is 6 mg/kg, the starting dose for the clinical trial was set at 6 mg/kg. The dose escalation study design is a 3 x 3 design, originally consisting of cohorts escalating by 3 mg/kg to 21 mg/kg (6, 9, 12, 15, 18 and 21 mg/kg). Since that time and based on safety data (see below), the FDA has permitted a revision to the study, in which the 12 and 15 mg/kg cohorts are skipped. Table 1 provides the original and current study design, with the corresponding total dose for a 70 kg patient. For example, 18 mg/kg is 1.26 g for a 70 kg patient. The trial also incorporates a validated Quality of Life questionnaire and a 12-month follow-up period. In Expanded Use, Single-Patient IND setting, 4 patients received at least one AVM0703 dose: glioblastoma: one 6 mg/kg;B-cell ALL: one 18 mg/kg dose;and two prostate cancer patients: one 18 mg/kg dose and patient #2: repeat doses for the past year as depicted in Table 2. Figure 1 depicts the flow cytometry analysis 24 hours following an 18 mg/kg AVM0703 dose. From a safety perspective, there have been no reports of drug-related or treatment emergent SAE's. The murine model safety findings correlate to the human experience. Adverse events reported to date have been self-limiting and mild to moderate. Frequent AEs include slight elevations of blood pressure, glucose and BUN that resolve without treatment in < 1 week post dose. Leukocytosis and lymphocytosis were reported 24 hours post infusion from the B-cell ALL patient but resolved by 7-days without reported intervention. Because a single AVM0703 dose triggers the rapid mobilization and activation of an endogenous bi-specific gamma-delta+ NKT cell with a favorable emerging safety profile, AVM0703 shows promise as a therapeutic agent in treating this serious disease. [Formula presented] Disclosures: Rea: AVM Biotechnology, LLC: Current Employment. Deisher: AVM Biotechnology, LLC: Current Employment. Jarzyna: AVM Biotechnology, LLC: Current Employment. Zahid: AVM Biotechnology, LLC: Ended employment in the past 24 months. Suwito: AVM Biotechnology, LLC: Current Employment. Poulin: AVM Biotechnology, LLC: Current Employment.

Interferon-Lambda Intranasal Protection and Differential Sex Pathology in a Murine Model of SARS-CoV-2 Infection.

Outbreaks of emerging viral pathogens like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are a major medical challenge. There is a pressing need for antivirals that can be rapidly deployed to curb infection and dissemination. We determined the efficacy of interferon lambda-1 (IFN-λ) as a broad-spectrum antiviral agent to inhibit SARS-CoV-2 infection and reduce pathology in a mouse model of disease. IFN-λ significantly limited SARS-CoV-2 production in primary human bronchial epithelial cells in culture. Pretreatment of human lung cells with IFN-λ completely blocked infectious virus production, and treatment with IFN-λ at the time of infection inhibited virus production more than 10-fold. To interrogate the protective effects of IFN-λ in response to SARS-CoV-2 infection, transgenic mice expressing the human angiotensin-converting enzyme 2 (ACE-2) were tested. One dose of IFN-λ administered intranasally was found to reduce animal morbidity and mortality. Our study with SARS-CoV-2 also revealed a sex differential in disease outcome. Male mice had higher mortality, reflecting the more severe symptoms and mortality found in male patients infected with SARS-CoV-2. The results indicate that IFN-λ potentially can treat early stages of SARS-CoV-2 infection and decrease pathology, and this murine model can be used to investigate the sex differential documented in COVID-19. IMPORTANCE The COVID-19 pandemic has claimed millions of lives worldwide. In this report, we used a preclinical mouse model to investigate the prophylactic and therapeutic value of intranasal IFN-λ for this acute respiratory disease. Specific vaccines have been responsible for curbing the transmission of SARS-CoV-2 in developed nations. However, vaccines require time to generate and keep pace with antigenic variants. There is a need for broad-spectrum prophylactic and therapeutic agents to combat new emerging viral pathogens. Our mouse model suggests IFN-λ has clinical utility, and it reflects the well-documented finding that male COVID-19 patients manifest more severe symptoms and mortality. Understanding this sex bias is critical for considering therapeutic approaches to COVID-19.

The SARS-CoV-2 spike protein subunit S1 induces COVID-19-like acute lung injury in Κ18-hACE2 transgenic mice and barrier dysfunction in human endothelial cells.

Acute lung injury (ALI) leading to acute respiratory distress syndrome is the major cause of COVID-19 lethality. Cell entry of SARS-CoV-2 occurs via the interaction between its surface spike protein (SP) and angiotensin-converting enzyme-2 (ACE2). It is unknown if the viral spike protein alone is capable of altering lung vascular permeability in the lungs or producing lung injury in vivo. To that end, we intratracheally instilled the S1 subunit of SARS-CoV-2 spike protein (S1SP) in K18-hACE2 transgenic mice that overexpress human ACE2 and examined signs of COVID-19-associated lung injury 72 h later. Controls included K18-hACE2 mice that received saline or the intact SP and wild-type (WT) mice that received S1SP. K18-hACE2 mice instilled with S1SP exhibited a decline in body weight, dramatically increased white blood cells and protein concentrations in bronchoalveolar lavage fluid (BALF), upregulation of multiple inflammatory cytokines in BALF and serum, histological evidence of lung injury, and activation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways in the lung. K18-hACE2 mice that received either saline or SP exhibited little or no evidence of lung injury. WT mice that received S1SP exhibited a milder form of COVID-19 symptoms, compared with the K18-hACE2 mice. Furthermore, S1SP, but not SP, decreased cultured human pulmonary microvascular transendothelial resistance (TER) and barrier function. This is the first demonstration of a COVID-19-like response by an essential virus-encoded protein by SARS-CoV-2 in vivo. This model of COVID-19-induced ALI may assist in the investigation of new therapeutic approaches for the management of COVID-19 and other coronaviruses.

Cytokine Signature Induced by SARS-CoV-2 Spike Protein in a Mouse Model.

Although COVID-19 has become a major challenge to global health, there are currently no efficacious agents for effective treatment. Cytokine storm syndrome (CSS) can lead to acute respiratory distress syndrome (ARDS), which contributes to most COVID-19 mortalities. Research points to interleukin 6 (IL-6) as a crucial signature of the cytokine storm, and the clinical use of the IL-6 inhibitor tocilizumab shows potential for treatment of COVID-19 patient. In this study, we challenged wild-type and adenovirus-5/human angiotensin-converting enzyme 2-expressing BALB/c mice with a combination of polyinosinic-polycytidylic acid and recombinant SARS-CoV-2 spike-extracellular domain protein. High levels of TNF-α and nearly 100 times increased IL-6 were detected at 6 h, but disappeared by 24 h in bronchoalveolar lavage fluid (BALF) following immunostimulant challenge. Lung injury observed by histopathologic changes and magnetic resonance imaging at 24 h indicated that increased TNF-α and IL-6 may initiate CSS in the lung, resulting in the continual production of inflammatory cytokines. We hypothesize that TNF-α and IL-6 may contribute to the occurrence of CSS in COVID-19. We also investigated multiple monoclonal antibodies (mAbs) and inhibitors for neutralizing the pro-inflammatory phenotype of COVID-19: mAbs against IL-1α, IL-6, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and inhibitors of p38 and JAK partially relieved CSS; mAbs against IL-6, TNF-α, and GM-CSF, and inhibitors of p38, extracellular signal-regulated kinase, and myeloperoxidase somewhat reduced neutrophilic alveolitis in the lung. This novel murine model opens a biologically safe, time-saving avenue for clarifying the mechanism of CSS/ARDS in COVID-19 and developing new therapeutic drugs.