GlyPerA™ effectively shields airway epithelia from SARS-CoV-2 infection and inflammatory events.

New SARS-CoV-2 variants of concern (VOCs) and waning immunity illustrate that quick and easy-to-use agents are needed to prevent infection. To protect from viral transmission and subsequent inflammatory reactions, we applied GlyperA™, a novel antimicrobial formulation that can be used as mouth gargling solution or as nasal spray, to highly differentiated human airway epithelia prior infection with Omicron VOCs BA.1 and BA.2. This formulation fully protected polarized human epithelium cultured in air-liquid interphase (ALI) from SARS-CoV-2-mediated tissue destruction and infection upon single application up to two days post infection. Moreover, inflammatory reactions induced by the Omicron VOCs were significantly lowered in tissue equivalents either pre-treated with the GlyperA™ solution, or even when added simultaneously. Thus, the GlyperA™ formulation significantly shielded epithelial integrity, successfully blocked infection with Omicron and release of viral particles, and decreased intracellular complement C3 activation within human airway epithelial cell cultures. Crucially, our in vitro data imply that GlyperA™ may be a simple tool to prevent from SARS-CoV-2 infection independent on the circulating variant via both, mouth and nose.

Antiviral drugs block replication of highly immune-evasive Omicron subvariants ex vivo, but fail to reduce tissue inflammation.

The identification of the SARS-CoV-2 Omicron variants BA.4/BA.5, BF.7 and BQ.1.1 immediately raised concerns regarding the efficacy of currently used monoclonal antibody therapies. Here we examined the activity of monoclonal antibody therapies and antiviral drugs against clinical specimens for SARS-CoV-2 Omicron BA.4/BA.5, BF.7 and BQ.1.1 employing an immunofluorescence neutralization assay. Further we explored treatment of BA.4/BA.5 infections with efficient antiviral drugs and monoclonal antibodies in a 3D model of primary human bronchial epithelial cells. We found that the antiviral drugs Molnupiravir, Nirmatrelvir and Remdesivir efficiently inhibit BA.4/BA.5, BF.7 and BQ.1.1 replication. In contrast, only the monoclonal antibody Cilgavimab exerted an inhibitory effect, while Tixagevimab, Regdanvimab and Sotrovimab lost their efficacy against BA.4/BA.5. We found that only the prophylactic treatment with Cilgavimab impacted on tissue inflammation by reducing intracellular complement component 3 (C3) activation following BA.4/BA.5 infection in primary human airway epithelial grown in air-liquid-interphase, which was not the case when using antiviral drugs or Cilgavimab after establishment of infection. Of note, all tested monoclonal antibodies had no neutralizing activity during infection by BF.7 and BQ.1.1 variants. Our results suggest that despite a marked reduction of viral replication, potent antiviral drugs fail to reduce tissue levels of inflammatory compounds such as C3, which can still result in tissue destruction.

Cilgavimab/Tixagevimab as alternative therapeutic approach for BA.2 infections.

Objectives: The identification of the SARS-CoV-2 Omicron variants BA.1 and BA.2 immediately raised concerns about the efficacy of currently used monoclonal antibody therapies. Here, we analyzed the activity of Sotrovimab and Regdanvimab, which are used in clinics for treatment of moderate to severe SARS-CoV-2 infections, and Cilgavimab/Tixagevimab, which are approved for prophylactic use, against BA.1 and BA.2 in a 3D model of primary human bronchial epithelial cells. Methods: Primary human airway epithelia (HAE) cells in a 3D tissue model were infected with clinical isolates of SARS-CoV-2 Delta, BA.1 or BA.2. To mimic the therapeutic use of mAbs, we added Regdanvimab, Sotrovimab or Cilgavimab/Tixagevimab 6 h after infection. In order to mirror the prophylactic use of Cilgavimab/Tixagevimab, we added this compound 6 h prior to infection to the fully differentiated, pseudostratified epithelia cultured in air-liquid interphase (ALI). Results: We observed that Sotrovimab, but not Regdanvimab, is active against BA.1; however, both antibodies lose their efficacy against BA.2. In contrast, we found that BA.2 was sensitive to neutralization by the approved prophylactic administration and the therapeutic use, which is not yet permitted, of Cilgavimab/Tixagevimab. Conclusion: Importantly, while the use of Tixagevimab/Cilgavimab is effective in controlling BA.2 but not BA.1 infection, monoclonal antibodies (mAbs) with efficacy against BA.1 are ineffective to reduce BA.2 virus replication in a human lung model. Our data may have implications on the variant specific clinical use of monoclonal antibodies.

Inhalation of Low Molecular Weight Heparins as Prophylaxis against SARS-CoV-2.

New SARS-CoV-2 variants of concern and waning immunity demonstrate the need for a quick and simple prophylactic agent to prevent infection. Low molecular weight heparins (LMWH) are potent inhibitors of SARS-CoV-2 binding and infection in vitro. The airways are a major route for infection and therefore inhaled LMWH could be a prophylactic treatment against SARS-CoV-2. We investigated the efficacy of in vivo inhalation of LMWH in humans to prevent SARS-CoV-2 attachment to nasal epithelial cells in a single-center, open-label intervention study. Volunteers received enoxaparin in the right and a placebo (NaCl 0.9%) in the left nostril using a nebulizer. After application, nasal epithelial cells were retrieved with a brush for ex-vivo exposure to either SARS-CoV-2 pseudovirus or an authentic SARS-CoV-2 isolate and virus attachment as determined. LMWH inhalation significantly reduced attachment of SARS-CoV-2 pseudovirus as well as authentic SARS-CoV-2 to human nasal cells. Moreover, in vivo inhalation was as efficient as in vitro LMWH application. Cell phenotyping revealed no differences between placebo and treatment groups and no adverse events were observed in the study participants. Our data strongly suggested that inhalation of LMWH was effective to prevent SARS-CoV-2 attachment and subsequent infection. LMWH is ubiquitously available, affordable, and easy to apply, making them suitable candidates for prophylactic treatment against SARS-CoV-2. IMPORTANCE New SARS-CoV-2 variants of concern and waning immunity demonstrate the need for a quick and simple agent to prevent infection. Low molecular weight heparins (LMWH) have been shown to inhibit SARS-CoV-2 in experimental settings. The airways are a major route for SARS-CoV-2 infection and inhaled LMWH could be a prophylactic treatment. We investigated the efficacy of inhalation of the LMWH enoxaparin in humans to prevent SARS-CoV-2 attachment because this is a prerequisite for infection. Volunteers received enoxaparin in the right and a placebo in the left nostril using a nebulizer. Subsequently, nasal epithelial cells were retrieved with a brush and exposed to SARS-CoV-2. LMWH inhalation significantly reduced the binding of SARS-Cov-2 to human nasal cells. Cell phenotyping revealed no differences between placebo and treatment groups and no adverse events were observed in the participants. Our data indicated that LMWH can be used to block SARS-CoV-2 attachment to nasal cells. LMWH was ubiquitously available, affordable, and easily applicable, making them excellent candidates for prophylactic treatment against SARS-CoV-2.

Cilgavimab/Tixagevimab as alternative therapeutic approach for BA.2 infections

Objectives The identification of the SARS-CoV-2 Omicron variants BA.1 and BA.2 immediately raised concerns about the efficacy of currently used monoclonal antibody therapies. Here, we analyzed the activity of Sotrovimab and Regdanvimab, which are used in clinics for treatment of moderate to severe SARS-CoV-2 infections, and Cilgavimab/Tixagevimab, which are approved for prophylactic use, against BA.1 and BA.2 in a 3D model of primary human bronchial epithelial cells. Methods Primary human airway epithelia (HAE) cells in a 3D tissue model were infected with clinical isolates of SARS-CoV-2 Delta, BA.1 or BA.2. To mimic the therapeutic use of mAbs, we added Regdanvimab, Sotrovimab or Cilgavimab/Tixagevimab 6 h after infection. In order to mirror the prophylactic use of Cilgavimab/Tixagevimab, we added this compound 6 h prior to infection to the fully differentiated, pseudostratified epithelia cultured in air-liquid interphase (ALI). Results We observed that Sotrovimab, but not Regdanvimab, is active against BA.1;however, both antibodies lose their efficacy against BA.2. In contrast, we found that BA.2 was sensitive to neutralization by the approved prophylactic administration and the therapeutic use, which is not yet permitted, of Cilgavimab/Tixagevimab. Conclusion Importantly, while the use of Tixagevimab/Cilgavimab is effective in controlling BA.2 but not BA.1 infection, monoclonal antibodies (mAbs) with efficacy against BA.1 are ineffective to reduce BA.2 virus replication in a human lung model. Our data may have implications on the variant specific clinical use of monoclonal antibodies.

Potent SARS-CoV-2-Specific T Cell Immunity and Low Anaphylatoxin Levels Correlate With Mild Disease Progression in COVID-19 Patients.

T cells play a fundamental role in the early control and clearance of many viral infections of the respiratory system. In SARS-CoV-2-infected individuals, lymphopenia with drastically reduced CD4+ and CD8+ T cells correlates with Coronavirus disease 2019 (COVID-19)-associated disease severity and mortality. In this study, we characterized cellular and humoral immune responses induced in patients with mild, severe and critical COVID-19. Peripheral blood mononuclear cells of 37 patients with mild, severe and critical COVID-19 and 10 healthy individuals were analyzed by IFNγ ELISpot and multi-color flow cytometry upon stimulation with peptide pools covering complete immunodominant SARS-CoV-2 matrix, nucleocapsid and spike proteins. In addition SARS-CoV-2 antibody levels, neutralization abilities and anaphylatoxin levels were evaluated by various commercially available ELISA platforms. Our data clearly demonstrates a significantly stronger induction of SARS-CoV-2 specific CD8+ T lymphocytes and higher IFNγ production in patients with mild compared to patients with severe or critical COVID-19. In all patients SARS-CoV-2-specific antibodies with similar neutralizing activity were detected, but highest titers of total IgGs were observed in critical patients. Finally, elevated anaphylatoxin C3a and C5a levels were identified in severe and critical COVID-19 patients probably caused by aberrant immune complex formation due to elevated antibody titers in these patients. Crucially, we provide a full picture of cellular and humoral immune responses of COVID-19 patients and prove that robust polyfunctional CD8+ T cell responses concomitant with low anaphylatoxin levels correlate with mild infections. In addition, our data indicates that high SARS-CoV-2 antibody titers are associated with severe disease progression.

C5aR inhibition of nonimmune cells suppresses inflammation and maintains epithelial integrity in SARS-CoV-2-infected primary human airway epithelia.

BACKGROUND: Excessive inflammation triggered by a hitherto undescribed mechanism is a hallmark of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and is associated with enhanced pathogenicity and mortality. OBJECTIVE: Complement hyperactivation promotes lung injury and was observed in patients suffering from Middle East respiratory syndrome-related coronavirus, SARS-CoV-1, and SARS-CoV-2 infections. Therefore, we investigated the very first interactions of primary human airway epithelial cells on exposure to SARS-CoV-2 in terms of complement component 3 (C3)-mediated effects. METHODS: For this, we used highly differentiated primary human 3-dimensional tissue models infected with SARS-CoV-2 patient isolates. On infection, viral load, viral infectivity, intracellular complement activation, inflammatory mechanisms, and tissue destruction were analyzed by real-time RT-PCR, high content screening, plaque assays, luminex analyses, and transepithelial electrical resistance measurements. RESULTS: Here, we show that primary normal human bronchial and small airway epithelial cells respond to SARS-CoV-2 infection by an inflated local C3 mobilization. SARS-CoV-2 infection resulted in exaggerated intracellular complement activation and destruction of the epithelial integrity in monolayer cultures of primary human airway cells and highly differentiated, pseudostratified, mucus-producing, ciliated respiratory tissue models. SARS-CoV-2-infected 3-dimensional cultures secreted significantly higher levels of C3a and the proinflammatory cytokines IL-6, monocyte chemoattractant protein 1, IL-1α, and RANTES. CONCLUSIONS: Crucially, we illustrate here for the first time that targeting the anaphylotoxin receptors C3a receptor and C5a receptor in nonimmune respiratory cells can prevent intrinsic lung inflammation and tissue damage. This opens up the exciting possibility in the treatment of COVID-19.