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

Abstract

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.

Keywords: SARS-CoV-2; infection prevention; low molecular weight heparin; virus-host interactions.

FIG 1

Flowchart of the volunteer inclusion. Volunteers were recruited within the AMC between January 7, 2021 and December 14, 2021. Both genders were eligible to participate. Inclusion criteria were an age between 18 and 65, good physical health (defined as not suffering from any illness or disease obstructing general daily functioning), and sufficient understanding of the Dutch language to comply to study procedures. Exclusion criteria were a positive SARS-CoV-2 antigen test (SARS-CoV-2 Antigen Rapid Test kit, JOYSBIO [Tianjin] Biotechnology Co. Ltd.), nasal-septum defects, usage of intranasal medication, frequent nosebleeds (>1/month), fever during the study visit (tympanic temperature >38.5°C during clinical visit), anamnestic or physical evidence of respiratory infection in the 4 weeks before the clinical visit, a known allergy or intolerance to LMWH or heparin-related products, a medical history of heparin-induced thrombocytopenia (HIT), the presence of mental disorders that would interfere with adherence to study procedures. Description of the inclusion process and parameters analysis, where 35 volunteers were assessed for eligibility, 1 volunteer withdrew before enrollment, and 34 volunteers were enrolled and received a placebo and 4500IE enoxaparin via nasal spray. Subsequently, the nasal epithelium was withdrawn and counted. One volunteer provided insufficient cellular material for all experiments and binding was subsequently not performed. Cells were plated and distributed into three categories for exposure to different viruses. A total of 19 donors provided material for pseudovirus binding. After the first 7 donors, we adapted the protocol due to the inefficient application of enoxaparin as measured by a tracer dye and these donors were excluded from the analysis. Next, 12 donors were included for analysis, 16 donors provided material for authentic SARS-CoV-2 virus binding, 3 donors were excluded from the analysis because the virus failed to bind in the placebo samples, and 13 donors were included for analysis. There was an overlap between donors, with 2 donors providing material for both SARS-CoV-2 pseudovirus and authentic virus binding.

FIG 2

SARS-CoV-2 binding and infection of polarized epithelial cells was blocked by LMWH enoxaparin. (A) SARS-CoV-2 pseudovirus binding was measured in polarized Caco-2 cells that were cultured in a static 3D model. The virus was either directly added or upon prior incubation with an in vitro enoxaparin (250 IU) control for 30 min. Binding with SARS-CoV-2 pseudovirus was measured using a p24 ELISA. (B) Polarized Caco-2 were infected with authentic SARS-CoV-2 (Italy; 10⁴ TCID₅₀) either without or in the presence of antibodies against ACE2 or after prior in vitro incubation with LMWH enoxaparin (250 IU) for 30 min. Authentic SARS-CoV-2 was detected after lysis by quantitative RT-PCR of viral RNA. (C) A 3D epithelial cell model of polarized normal human bronchial epithelial cells was treated with enoxaparin (nebulization) and SARS-CoV-2 was added at different time points (0.5 h, 4 h, 16 h, and 24 h). SARS-CoV-2 infection was measured by quantitative RT-PCR of viral RNA after 24 h. The data show the mean values and error bars are the SEM. Statistical analysis was performed using (A) one-way ANOVA with Tukey’s multiple-comparison test; *, P = 0.0203; ***, P = 0.0005; ****, P < 0.0001 (n = 2), or (B) one-way ANOVA with Tukey’s multiple-comparison test; ****, P < 0.0001 (n = 2). (C) n = 2.

FIG 3

Characterization of primary human nasal epithelial cells. (A) Flow cytometry analysis of single-cell suspensions from the nasal epithelium. (A and B) Primary human nasal epithelial cells isolated from the nasal mucosa of healthy volunteers after treatment with either placebo (saline solution) or LMWH enoxaparin were directly labeled with pan-cytokeratin (Pan-C) and CD45. Additionally, cells were labeled with antibodies against ACE2 and heparan sulfate (HS) (n = 17). (C) Cells from the LMWH enoxaparin-treated nostril were additionally stained with antibodies against the surface markers EpCAM, Muc-5B, CD3, CD11b, and CD11c (n = 17, 4 donors excluded; CD3 n = 8). (D) Cells from both the placebo and LMWH enoxaparin-treated nose were stained for Pan-C, CD45, ACE2, HS, EpCAM, Muc-5B, CD3, CD11b and CD11c (n = 9, 4 donors excluded). The cellular phenotype was monitored using flow cytometry analysis. ns, not significant.

FIG 4

In vivo enoxaparin inhalation prevented SARS-CoV-2 pseudovirus from binding to nasal epithelial cells. (A) Nasal epithelial cells isolated from the placebo-treated volunteers (either unvaccinated [white circle, n = 6], partially vaccinated [white/black circle, n-1], or fully vaccinated [black circle, n = 12]) were exposed to SARS-CoV-2 pseudovirus. Binding was measured after 4 h by ELISA. (B and C) Nasal epithelial cells isolated from the nostrils of volunteers were treated with either a placebo or LMWH enoxaparin and were exposed to SARS-CoV-2 pseudovirus (D) in the presence or absence of an additional in vitro enoxaparin condition (250 IU) and binding was measured after 4 h by ELISA. Data show the mean values and error bars are the SEM. Statistical analysis was performed using (B) a two-way ANOVA with Tukey’s multiple-comparison test; ****, P < 0.0001 (n = 12), (C) a two-tailed, unpaired, nonparametric, Mann-Whitney test; **, P = 0.0043 (n = 12); or (D) a two-way ANOVA with Tukey’s multiple-comparison test; ****, P < 0.0001; ns, not significant (n = 12).

FIG 5

In vivo enoxaparin inhalation prevented authentic SARS-CoV-2 binding to nasal epithelial cells. (A and B) Nasal epithelial cells retrieved from the nasal cavity of healthy volunteers after exposure to placebo (saline solution, left nostril) and LMWH enoxaparin (right nostril) were exposed to authentic SARS-CoV-2 (hCOV-19 Italy) for 4 h at 4C compared to an uninfected control sample (A). (C) Additionally, authentic SARS-CoV-2 was incubated in the presence or absence of in vitro LMWH enoxaparin (250 IU) for 30 min before inoculation of epithelial cells. (A to C) Virus binding was measured after 4 h by real-time RT-PCR. Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A) a two-way ANOVA with Tukey’s multiple-comparison test; **, P = 0.0036; *, P = 0.0315 (n = 12); or (B) a two-tailed, unpaired, nonparametric, Mann-Whitney test; **, P = 0.0387; ns, not significant.