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Principal Investigator: EDWARD D CHAN
Organization: VA EASTERN COLORADO HEALTH CARE SYSTEM
Fiscal Year: 2024
Funding agency: Veterans Affairs
The clinical outcome for patients with severe COVID-19 remains poor due to the lack of highly efficacious
treatment for such individuals. Finding a better remedy for them is an important niche and immediate unmet
need. The aim of this pre-clinical project is to substantiate the therapeutic effect of combined alpha-1-
antitrypsin (AAT) + enoxaparin (a low molecular weight heparin) against SARS-CoV-2 infection and its
consequences. Establishing the efficacy of AAT + enoxaparin combination will provide a necessary foundation
for future clinical trials with the goal of employing effective therapy for those with severe COVID-19.
We have novel biological evidence supported by Artificial Intelligence-based molecular modeling
that enoxaparin synergizes with AAT to inhibit TMPRSS2 (a cell surface protease that activates the spike
protein of SARS-CoV-2) and to reduce SARS-CoV-2 burden in primary human airway epithelial cells (hAEc)
and monocyte-derived macrophages (MDM). Because both AAT and enoxaparin embrace a panoply of
activities that antagonize other pathogenic mechanisms of severe COVID-19 – including anti-inflammatory,
anti-thrombotic, pro-autophagy (known to kill MERS-CoV), and endothelial cell protection – we hypothesize
that the AAT + enoxaparin combination will be most effective (compared to each alone) in mitigating SARS-
CoV-2 infection and its consequences.
We will use three complementary models to elucidate the efficacy of AAT, enoxaparin, and combination of
both against SARS-CoV-2 infection: (i) primary hAEc since they express high ACE2 levels, fulminant airway
disease occurs, and infection of ciliated hAEc and breach of their defense initiates a portal of entry into the
lower airways / alveoli to cause COVID-19 pneumonia; (ii) MDM + plasma derived from AAT-deficient
individuals immediately before and immediately after receiving routine intravenous AAT since macrophages
are key orchestrators of the hyper-inflammatory response seen with COVID-19; and (iii) two murine models,
one with wildtype AAT and another with AAT knocked out.
Aim 1: Determine in primary hAEc the mechanisms by which AAT, enoxaparin, and both reduce SARS-
CoV-2 infection and its consequences. Approach: hAEc will be infected with SARS-CoV-2 followed by no
treatment or treatment with AAT, enoxaparin, or combination of both and assayed for viral load, autophagic
flux, pro-inflammatory cytokines, and hAEc viability and barrier integrity.
Aim 2: Determine in macrophages the mechanisms by which AAT (given in vivo) ± enoxaparin mitigate
SARS-CoV-2 infection. Approach: infect human MDM cultured in autologous plasma – prepared from AAT-
deficient individuals before and after AAT infusions ± ex vivo enoxaparin – with SARS-CoV-2 and determine
viral load, autophagic flux, and pro-inflammatory cytokine / macrophage extracellular trap (METs) production.
Aim 3: Determine if SARS-CoV-2 infection of mice is mitigated by AAT, enoxaparin, and combination of
both. Approach: transgenic (Tg) mice bred to express human ACE2 with or without deletion of AAT (to
represent humans who are AAT-replete and AAT-deficient, respectively) will either be left untreated or treated
with AAT, enoxaparin, or the combination along with SARS-CoV-2 infection. From the lungs and spleens, we
will quantify viral load and the phenotypes of macrophages, dendritic cells, CD4+ T cells, and CD8+ T cells in
both organs, as well as analyze the lungs for epithelial and endothelial injury, co-localization of SARS-CoV-2
with airway and alveolar epithelial cells, and neutrophil extracelluar trap (NETs) formation (both METs and
NETs implicated in immunothrombosis of severe COVID-19).
Establishing the efficacy of AAT + enoxaparin combination will provide a foundation for future clinical trials
with the goal of employing more effective therapy for veterans and non-veterans with severe COVID-19.
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procedures><Injury><Intravenous><Knock-out><Knockout><LMWH><Left><Lovenox><Low-Molecular-Weight Heparin><Lung><Lung Alveolar Epithelia><Lung Respiratory System><Lytotoxicity><MERS corona virus><MERS coronavirus><MERS virus><MERS-CoV><Machine Intelligence><Macrophage><Marrow Neutrophil><Marrow monocyte><Mice><Mice Mammals><Middle East Respiratory Syndrome Corona Virus><Middle East Respiratory Syndrome Coronavirus><Middle East Respiratory Syndrome Virus><Middle East Respiratory Syndrome-CoV><Middle East Respiratory Virus><Middle East Respiratory coronavirus><Middle Eastern Respiratory Syndrome Corona virus><Middle Eastern Respiratory Syndrome Coronavirus><Middle Eastern Respiratory Syndrome Virus><Middle Eastern Respiratory Syndrome-CoV><Modeling><Modern Man><Molecular Modeling Nucleic Acid Biochemistry><Molecular Modeling Protein/Amino Acid Biochemistry><Molecular Models><Murine><Mus><Mφ><Neutrophilic Granulocyte><Neutrophilic Leukocyte><Organ><PRSS10><Pathogenicity><Patient outcome><Patient-Centered Outcomes><Patient-Focused Outcomes><Peptidases><Peptide Hydrolases><Phenotype><Plasma><Plasma Serum><Polymorphonuclear Cell><Polymorphonuclear Leukocytes><Polymorphonuclear Neutrophils><Pre-Clinical Model><Preclinical Models><Production><Property><Protease Gene><Proteases><Proteinases><Proteins><Proteolytic Enzymes><Respiratory Epithelium><Reticuloendothelial System, Serum, Plasma><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 S><SARS-CoV-2 S protein><SARS-CoV-2 associated pneumonia><SARS-CoV-2 burden><SARS-CoV-2 disease burden><SARS-CoV-2 induced pneumonia><SARS-CoV-2 infection><SARS-CoV-2 infection burden><SARS-CoV-2 inhibitor><SARS-CoV-2 pneumonia><SARS-CoV-2 related pneumonia><SARS-CoV-2 spike><SARS-CoV-2 spike glycoprotein><SARS-CoV-2 spike protein><SARS-CoV-2 viral pneumonia><SARS-CoV2><SARS-CoV2 infection><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Safety><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 S protein><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome coronavirus 2 inhibitor><Severe acute respiratory syndrome coronavirus 2 spike glycoprotein><Severe acute respiratory syndrome coronavirus 2 spike protein><Severe acute respiratory syndrome related corona virus 2><Spleen><Spleen Reticuloendothelial System><Structure of respiratory epithelium><T4 Cells><T4 Lymphocytes><T8 Cells><T8 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deficiency><α1-Antitrypsin><α1-Antitrypsin Deficiency><α1-Proteinase Inhibitor>