Crosstalk between TLR8 and RIG-I-like receptors enhances antiviral immune responses.

Background: Toll-like receptor (TLR) agonists have been investigated due to their potential dual effects as latency reverting agents and immune modulatory compounds in people living with HIV (PLWH). Here, we investigated whether co-stimulation of TLR7/8 agonists with RIG-I-like receptor (RLR) agonists enhances antiviral immunity. Methods: Peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (DCs) were incubated with TLR and RLR-agonists for 24 h and innate and adaptive immune responses were determined (maturation markers, cytokines in supernatant, ISG expression). Results: Both TLR7 and TLR8 agonists induced pro-inflammatory cytokines in DCs as well as PBMCs. TLR8 agonists were more potent in inducing cytokine responses and had a stronger effect on DC-induced immunity. Notably, while all compounds induced IL-12p70, co-stimulation with TLR8 agonists and RLR agonist polyI: C induced significantly higher levels of IL-12p70 in PBMCs. Moreover, crosstalk between TLR8 and RLR agonists induced a strong type I Interferon (IFN) response as different antiviral IFN-stimulated genes were upregulated by the combination compared to the agonists alone. Conclusion: Our data strongly suggest that TLR crosstalk with RLRs leads to strong antiviral immunity as shown by induction of IL-12 and type I IFN responses in contrast to TLRs alone. Thus, co-stimulation of TLRs and RLRs might be a powerful strategy to induce reactivation of latent reservoir as well as antiviral immunity that eliminates the reactivated cells.

Low energy nebulization preserves integrity of SARS-CoV-2 mRNA vaccines for respiratory delivery.

Nebulization of mRNA therapeutics can be used to directly target the respiratory tract. A promising prospect is that mucosal administration of lipid nanoparticle (LNP)-based mRNA vaccines may lead to a more efficient protection against respiratory viruses. However, the nebulization process can rupture the LNP vehicles and degrade the mRNA molecules inside. Here we present a novel nebulization method able to preserve substantially the integrity of vaccines, as tested with two SARS-CoV-2 mRNA vaccines. We compare the new method with well-known nebulization methods used for medical respiratory applications. We find that a lower energy level in generating LNP droplets using the new nebulization method helps safeguard the integrity of the LNP and vaccine. By comparing nebulization techniques with different energy dissipation levels we find that LNPs and mRNAs can be kept largely intact if the energy dissipation remains below a threshold value, for LNP integrity 5-10 J/g and for mRNA integrity 10-20 J/g for both vaccines.

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.

Infection and transmission of SARS-CoV-2 depend on heparan sulfate proteoglycans.

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and outbreaks of new variants highlight the need for preventive treatments. Here, we identified heparan sulfate proteoglycans as attachment receptors for SARS-CoV-2. Notably, neutralizing antibodies against SARS-CoV-2 isolated from COVID-19 patients interfered with SARS-CoV-2 binding to heparan sulfate proteoglycans, which might be an additional mechanism of antibodies to neutralize infection. SARS-CoV-2 binding to and infection of epithelial cells was blocked by low molecular weight heparins (LMWH). Although dendritic cells (DCs) and mucosal Langerhans cells (LCs) were not infected by SARS-CoV-2, both DC subsets efficiently captured SARS-CoV-2 via heparan sulfate proteoglycans and transmitted the virus to ACE2-positive cells. Notably, human primary nasal cells were infected by SARS-CoV-2, and infection was blocked by pre-treatment with LMWH. These data strongly suggest that heparan sulfate proteoglycans are important attachment receptors facilitating infection and transmission, and support the use of LMWH as prophylaxis against SARS-CoV-2 infection.