On December 31, 2019, a group of cases of pneumonia of unknown etiology was reported in Wuhan, China. On January 10, 2020, a new coronavirus was identified as the pathogen, named SARS-CoV-2, and classified in the genus Betacoronavirus. On April 20, 2020, 5 weeks after the world health organization declared COVID-19 a pandemic, the new COVID-19 coronavirus caused more than 166,000 deaths among 2.5 million confirmed cases reported in at least 185 countries or territories worldwide.
The new SARS-CoV-2 coronavirus has identical genomic sequences with the previous SARS-CoV and MERS-CoV coronaviruses ≈79% and ≈50%, respectively. All three viruses differ in terms of epidemiology and physiopathology.
There is currently no specific antiviral treatment or vaccine for any of these three coronaviruses. Standard patient management is based primarily on symptom monitoring and respiratory support when needed.
To study the features of COVID-19, we need biologically relevant preclinical experimental models of SARS-CoV-2 infection as a complement to the Vero E6 African green monkey cell line.
Previously, it was reported about the advantage of using restored human respiratory epithelium (HAE) as a physiologically relevant model for isolation, culture, and study of a wide range of respiratory viruses. Based on biopsies of nasal or bronchial cells differentiated in the air-liquid interphase, these models reproduce most of the main structural, functional, and innate immune features of the human respiratory epithelium with high accuracy. These features play a central role in the early stages of infection and are reliable surrogates for studying the mechanisms of respiratory diseases and drug discovery.
The study initially isolated the SARS-CoV-2 virus from a nasal swab from one of the first hospitalized patients with confirmed COVID-19 in France. The number of copies of viral DNA was increased in cells of the African green APE Vero E6. After that, restored epithelial models of the human respiratory tract (HAE) of nasal and bronchial origin were used to study the kinetics of SARS-CoV-2 and its induced tissue remodelling of cellular ultrastructure and transcriptional immune signatures, and to evaluate the therapeutic potential of COVID-19 combination therapy.
Isolation and characterization of SARS-CoV-2 in Vero E6 and HAE cells
The complete genome sequence of the isolated SARS-CoV-2 virus is deposited in the GISAID EPic.oVTM database at the link BetaCoV/France/IDF0571/2020 (access ID EPI_ISL_411218). Phylogenetic analysis confirmed that the isolated virus is representative of the currently circulating strains.
The replication capacity of SARS-CoV-2 in Vero E6 cells was studied in various infection multiplicity (MOI) (Pic. 1A) using both the classical determination of infectious titer in cell culture (TCID50) and molecular semi-quantitative methods based on specific ORF1b-nsp14 primers and probes developed by the school of public health of the University of Hong Kong. This two-way approach showed an observed cytopathic effect 48 hours after infection (hpi) (Pic. 1B) and allowed to test a large interval (range 1-8 log10 (TCID50)) with a high correlation (R2 0.94) between molecular and infectious viral titers (Pic. 1C).
Pic. 1. Characteristics of the SARS-CoV-2 infectious model in Vero E6 cells, as well as in restored nasal and bronchial HAE
During the study, nasal mucus obtained from a nasal smear (MucilAir HAE) was inoculated on the aPic.al surface of HAE, which was confirmed by observations of transmission electron microscopy. High accumulation of hereditary virions in goblet-like mucus-producing cells was easily discernible on both the aPic.al and basal sides of HAE through 48 hpi.
Then, the MucilAir HAE model and protocols previously optimized for various respiratory viruses were used for experimental SARS-CoV-2 infection. Virus replication was controlled by repeated sampling and titration of TCID50 on the aPic.al surface of HAE (Pic. 1D). Transepithelial electrical resistance (TEER), which is considered a surrogate for epithelial integrity, was also measured during infection (Pic. 1E). In parallel, a comparative quantitative determination of the molecular viral genome was performed at three levels of the air-liquid interphase HAE: in aPic.al washes (Pic. 1F, aPic.al), total cellular RNA (Pic. 1G, intracellular) and basal medium (Pic. 1H, basal).
SARS-CoV-2 virus production on the aPic.al surface of the epithelium increased sharply after 48 hpi, with the highest virus titers (>7 log10 TCID50/ml) observed after 72-96 hpi (Pic. 1D and 1F). A sharp increase in viral replication correlated with a decrease in epithelial integrity after 48 hpi, which was reflected by >2.8-and 4-fold decrease in the values of TEER HAE of the bronchi and nose, respectively, with subsequent partial recovery in the case of bronchial HAE (Pic. 1E).
Besides, viral production at the aPic.al pole correlated well with intracellular detection of the viral genome via 48 hpi, except for nasal HAE. A robust relative increase in nsp14 RNA was observed in nasal HAE (Pic. 1F). The viral genome was detected in the basal environment after 48 hpi and further (Pic. 1H), which indirectly confirms the violation of epithelial integrity caused by infection after 48 hpi and previously detected using TEER measurements.
SARS-CoV-2 induces remodelling of HAE cell ultrastructure
To further characterize the biology of SARS-CoV-2, both nasal (Pic. 2A and 2B) and bronchial (Pic. 2C and 2D) HAE were inoculated. Infection-induced cell ultrastructure remodelling was analyzed using transmission electron microscopy.
After 48 hpi, ciliated, goblet-shaped, and to a lesser extent, basal cells of both HAES showed active production of viral offspring. This observation is consistent with the results of viral replication described in Picture 1, as well as with a recent study that reported high levels of angiotensin-converting enzyme SARS-CoV-2 (ACE2) expression in both ciliated and goblet-shaped respiratory cells.
As previously observed in structural studies of other coronaviruses, in particular SARS-CoV and MERS-CoV, the study identified characteristic clusters in the perinuclear region of infected HAE cells. These clusters mainly consist of numerous viral one-and two-membrane vesicles (DMV) and mitochondria (Pic. 2A, 2A1, 2B, 2C, and 2D). Large electron-dense structures corresponding to the accumulation of viral material in the areas of active replication of the virus, as well as tyPic.al two-membrane spherules containing fragments of membranes and located between the formed virions, were also observed at 48 hpi (Pic. 2B and 2D). Also, near plasma membranes, spherules with a double membrane containing numerous virions are noticeable (Pic. 2B1 and 2D2). These spherules, as well as several clusters of virions, were observed mainly on the surface of ciliated cells (Pic. 2A2). These features are tyPic.al for the later stages of the viral cycle, which confirms the ability of HAE to reproduce the asynchronous nature of the infection.
Picture 2. Ultrastructure of infected SARS-CoV-2 restored nasal and bronchial HAE
SARS-CoV-2 induces early immune responses in nasal and bronchial HAE
Recent studies have linked COVID-19 to high plasma levels of specific immunostimulating and Pro-inflammatory cytokines (such as IL6), especially in patients with severe disease, suggesting a potential association with a poor prognosis. However, until now, this inflammatory condition was much less tyPic.al for the respiratory microenvironment.
To investigate the effect of SARS-CoV-2 infection on gene expression, we use nanostring hybridization technology on nasal and bronchial HAE to multiplex mRNA detection and relative quantification of two complementary panels of genes involved in the immune response.
The heat map and hierarchical analysis revealed two different levels of clustering. Regardless of the nasal or bronchial nature of the HAE model, ∼14% of the studied genes have a noticeable increase in expression after 24 hpi, which subsequently decreases after 48, 72 and 96 hpi (Pic. 3A). This observation was confirmed by independent data analysis using a complete gene panel. The first component of the principal component analysis (PCA), which accounts for 63% of the variance, is mainly determined by the time of infection, with a clear difference between 24 hpi and other time points (Pic. 3B, red triangles/dots).
Immune transcriptomic signatures appear to be partly due to the nature of HAE after 24 hpi (Picture 3A). It is consistent with the second component analysis, which collected 12.6% of the total variance, which allowed for a clear differentiation between the nasal and bronchial divisions (Pic. 3B, green/purple triangles and dots).
According to a recent report by Blanco-Melo and his collaborators, interferon (IFN) and interferon-stimulated gene (ISG) responses were almost undetectable in the first 24 hpi. However, the signature of innate immune expression after 24 hpi (peak at 72-96 hpi) is due to strong activation of IFN types I and III (IFNB1, IFNL1 and IFNL2, -3 and -4), as well as immunity-related genes, especially in nasal HAE.
Only a subset of these genes (CXCL10/IP10, CXCL2/MIP2A, IL1A, IL1B, Mx1, and ZBP1) follow the same pattern in bronchial HAE, although, at an overall lower expression level, the first modulation of IFNB1, IFNL1, CCL2/MCP1, and IL-6 in this tissue appears to disappear after 96 hpi. Besides, the relative expression of a subset of genes associated with nuclear factor-kB (NF-kB) and tumour necrosis factor α (TNF-α) (for example, IL-18, IL-18R1, NFKB2, NFKBIA, TNFA, and TNFAIP3) remains mostly unchanged throughout infection in bronchial HAE, but increases sharply in nasal HAE after 48 hpi and beyond (Picture 3C).
The results highlight the distinctive transcriptional immune signatures between nasal and bronchial HAE both in terms of kinetics and intensity, suggesting potential internal differences in the early response to SARS-CoV-2 infection between the upper and lower respiratory tracts. These results are consistent with the first clinical reports describing in some patients a rapid deterioration of respiratory status and general clinical condition on the 7th-10th day after the onset of symptoms, which is most likely associates with cytokine storm syndrome.
Pic. 3. nasal and bronchial innate immune transcription signature during SARS-CoV-2
Combined treatment with Redecision and Diltiazem enhances the effectiveness of monotherapy with Redecision
Currently, there are no specific treatments for COVID-19. Therefore, approved and experimental drugs intended for the treatment of other diseases are repurposed as medicines. These treatments are usually based on limited clinical or preclinical data.
Remdesivir (GS-5734) is a prodrug of an adenosine nucleotide analogue with demonstrated broad antiviral activity against several RNA viruses in various preclinical models. Recently, Remdesivir has shown promising results in animal models for the treatment of various coronaviruses, including MERS-CoV, as well as in one in vitro study against SARS-CoV-2. Clinical trials on Remdesivir for the COVID-19 treatment have already begun in China and the United States.
Diltiazem is a Ca2 + channel antagonist commonly used as an antihypertensive to control angina and cardiac arrhythmia. In a recent study, Diltiazem was repurposed as an effective host-directed flu inhibitor. The antiviral effect of Diltiazem is based on its hitherto undescribed ability to induce an antiviral IFN response, especially IFN-1β and type III IFNs. A phase IIb clinical trial is currently underway to evaluate the effectiveness of combination therapy with Diltiazem and Oseltamivir in patients with severe influenza (FLUNEXT TRIAL PHRC #15-0442, NCT03212716).
The rationale for testing such a combination of drugs targeting both the virus and the host is consistent with an assessment of the potential benefits of combined COVID-19 treatment with Remdesivir and Diltiazem. This strategy is also supported by a recent study describing hypertension as a risk factor among inpatient patients with COVID-19, as well as two reports that do not anticipate potential side effects of Diltiazem or negative pharmacological interaction between Remdesivir and Diltiazem for the treatment of COVID-19.
The study examined the antiviral potential of monotherapy with Remdesivir, as well as in combination with Diltiazem, both Vero E6 and HAE. Picture 4A-4C shows a robust antiviral effect of post-infection treatment with Remdesivir in Vero E6 cells with 50% inhibitory concentration values (IC50) of 0.98 ± 0.07 µm after 48 hpi and 0.72 ± 0.03 µm after 72 hpi and a selectivity index (SI) of 281 and 347, respectively.
Although Vero E6 cells produce IFN-λ1, this cell line cannot produce type I IFN. This incomplete IFN response most likely explains the lack of significant antiviral effect observed with Diltiazem monotherapy under experimental conditions. However, the addition of 11.5 µm Diltiazem significantly increased the antiviral effect of Ramdevpir. That is evidenced by a 3-and 2-fold decrease in IC50 values compared to Remdesivir monotherapy at 48 and 72 hpi, respectively (dose-response curves in Pic. 4A-4C).
Picture 4. Evaluation of the antiviral activity of the combination of Remdesivir and Diltiazem in Vero E6 and HAE cells.
Daily basolateral treatment of HAE with Remdesivir 1.25, 5, and 20 microns resulted in a decrease in intracellular titers of the SARS-CoV-2 nasal HAE virus by 3.5, 3.1, and 7.3 log10 after 48 hpi, respectively. A decrease in the bronchial HAE virus titers by 7.0, 5.8, and 7.9 log10 were observed at the same time (Pic. 4D, upper panel). Nasal and bronchial viral titers also decreased 72 hpi after treatment with 1.25 microns (6.9 and 7.0 log10), 5 microns (8.0 and 8.3 log10), and 20 microns (2.4 and 2.0 log10) of Remdesivir, respectively ( Picture 4E, upper panel).
For a model with a full functional IFN response, it is not surprising that daily treatment with Diltiazem 90 microns resulted in a moderate decrease in intracellular viral titers in nasal (40% and 69%) and bronchial (80% and 24%) HAE through 48 and 72 hpi, respectively (Pic. 4D and 4E, upper panel).
The study observed an additional decrease in viral titers in nasal HAE by 1.45 and 1.3 log10 after 48 hpi for the Remdesivir-Diltiazem combination compared to Remdesivir monotherapy of 5 and 20 microns, respectively, although only the former was statistically significant (P = 0.0066).
Also, TEER analysis showed that antiviral effects induced by Remdesivir, Diltiazem, or a combination of Remdesivir-Diltiazem are mainly expressed in protecting the integrity of the nasal epithelial barrier through 48 and 72 hpi (Pictures 4D and 4E, bottom panels).
Regardless of its effect on reducing viral activity, the three tested combinations of Remdesivir-Diltiazem were particularly useful in protecting the integrity of bronchial HAE through 72 hpi. That was proved by TEER values comparable to those of uninfected control groups (Pic. 4E, bottom panel).
Study limitations
The absence of white blood cells in the HAE model (for example, neutrophils, monocytes, dendritic cells) may partially distort the analysis of the innate immune response induced by infection. Although this study demonstrates the potential of the Remdesivir-Diltiazem combination in the treatment of SARS-CoV-2, its verification and implementation in clinical practice need further study.
Conclusions
The combination of a drug directed against the virus and a drug directed at the host can lead to increased antiviral and immunomodulatory effects, including the immunopathological phase, often observed during the second week of infection. This combination can also reduce the therapeutic doses of drugs that target nucleic acid synthesis, and therefore minimize the expected side effects.
