Saenkham-Huntsinger, P. | Search bioRxiv

bioRxiv2023

Characterization of Unique Pathological Features of COVID-Associated Coagulopathy: Studies with AC70 hACE2 Transgenic Mice Highly Permissive to SARS-CoV-2 Infection

COVID-associated coagulopathy seemly plays a key role in post-acute sequelae of SARS-CoV-2 infection. However, the underlying pathophysiological mechanisms are poorly understood, largely due to the lack of suitable animal models that recapitulate key clinical and pathological symptoms. Here, we fully characterized AC70 line of human ACE2 transgenic (AC70 hACE2 Tg) mice for SARS-CoV-2 infection. We noted that this model is highly permissive to SARS-CoV-2 with values of 50% lethal dose and infectious dose as [~] 3 and [~] 0.5 TCID50 of SARS-CoV-2, respectively. Mice infected with 105 TCID50 of SARS-CoV-2 rapidly succumbed to infection with 100% mortality within 5 days. Lung and brain were the prime tissues harboring high viral titers, accompanied by histopathology. However, viral RNA and many inflammatory mediators could be readily detectable in other organs, suggesting the nature of a systemic infection. Lethal challenge of AC70 hACE2 Tg mice caused acute onset of leukopenia, lymphopenia, along with an increased neutrophil-to-lymphocyte ratio. Importantly, infected animals recapitulated key features of COVID-19-associated coagulopathy, including significantly elevated levels of D-dimer, t-PA, PAI-1, and circulating NETs, along with activated platelet/endothelium marker. Immunohistochemical staining with anti-PF4 antibody revealed profound platelet aggregates especially within blocked veins of the lungs. ANXA2 is known to interact with S100A10 to form heterotetrametric complexes, serving as coreceptors for t-PA to regulate membrane fibrinolysis. Thus, our results revealing elevated IgG type anti-ANXA2 antibody production, downregulated de novo ANXA2/S100A10 synthesis, and reduced AnxA2/S100A10 association in infected mice support an important role of this protein in the pathogenesis of acute COVID-19. In summary, we showed that acute SARS-CoV-2 infection of AC70 hACE2 Tg mice triggered a hypercoagulable state coexisting with ill-regulated fibrinolysis, accompanied by dysregulation of ANXA2 system, which might serve as druggable targets for development of antithrombotic and/or anti-fibrinolytic agents to attenuate pathogenesis of COVID-19. Author SummaryAccumulating evidence strongly suggests that COVID-associated coagulopathy characterized by dysregulation of the coagulation cascade, fibrinolysis system and pulmonary microvascular immune-thrombosis during different stages of SARS-CoV-2 infection may have a "yet-to-be fully defined" impact on the development of post-acute sequela of COVID-19. Herein we initially reported a comprehensively characterized AC70 hACE2 Tg mouse model for SARS-CoV-2 infection and disease. We next demonstrated the subsequent onset of imbalanced coagulation and fibrinolysis pathways in infected Tg mice, focusing on dysregulated formation of ANXA2/S100A10 complexes, key coreceptors for t-PA that regulates membrane fibrinolysis, in which elevated production of autoantibodies against ANXA2 induced by SARS-CoV-2 might play an intriguing role. Taken together, we demonstrated that AC70 hACE2 Tg mice lethally challenged with SARS-CoV-2 recapitulated several features of COVID-associated coagulopathy observed in patients and highlighted the potential role of ANXA2 in this phenomenon. Thus, ANXA2 might serve as a potentially novel druggable target to attenuate COVID-19-associated thrombotic events.

microbiology↗

bioRxiv2023

Design of SARS-CoV-2 protease inhibitors with improved affinity and reduced sensitivity to mutations

Inhibitors of the SARS-CoV-2 main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function on these mutants are thus urgently needed. We hypothesized that the covalent HCV protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more tightly than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to NTV. A crucial feature of ML2006a4 is a novel derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Finally, ML2006a4 is less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory drug design can preemptively address potential resistance mechanisms.

microbiology↗

bioRxiv2022

A pan-variant mRNA-LNP T cell vaccine protects HLA transgenic mice from mortality after infection with SARS-CoV-2 Beta

Clinically licensed COVID-19 vaccines ameliorate viral infection by inducing vaccinee production of neutralizing antibodies that bind to the SARS-CoV-2 Spike protein to inhibit viral cellular entry (Walsh et al., 2020; Baden et al., 2021), however the clinical effectiveness of these vaccines is transitory as viral variants arise that escape antibody neutralization (Tregoning et al., 2021; Willett et al., 2022). Vaccines that solely rely upon a T cell response to combat viral infection could be transformational because they can be based on highly conserved short peptide epitopes that hold the potential for pan-variant immunity, but a mRNA-LNP T cell vaccine has not been shown to be sufficient for effective antiviral prophylaxis. Here we show that a mRNA-LNP vaccine based on highly conserved short peptide epitopes activates a CD8+ and CD4+ T cell response that prevents mortality in HLA-A*02:01 transgenic mice infected with the SARS-CoV-2 Beta variant of concern (B.1.351). In mice vaccinated with the T cell vaccine, 24% of the nucleated cells in lung were CD8+ T cells on day 7 post infection. This was 5.5 times more CD8+ T cell infiltration of the lungs in response to infection compared to the Pfizer-BioNTech Comirnaty(R) vaccine. Between days 2 and 7 post infection, the number of CD8+ T cells in the lung increased in mice vaccinated with the T cell vaccine and decreased in mice vaccinated with Comirnaty(R). The T cell vaccine did not produce neutralizing antibodies, and thus our results demonstrate that SARS-CoV-2 viral infection can be controlled by a T cell response alone. Our results suggest that further study is merited for pan-variant T cell vaccines, and that T cell vaccines may be relevant for individuals that cannot produce neutralizing antibodies or to help mitigate Long COVID.

immunology↗