Old Paper (Therapeutic): A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike

Questions to answer about each paper:

Please provide 1-2 sentences introducing the study and its main findings

In this study, Shuai Xia et al. evaluate the inhibitory potential of their in-house OC43-HR2P-modified peptide EK1 against various human coronaviruses. EK1 showed significant viral inhibition in vivo and in vitro.

Study question(s) being investigated:

How many/what drugs/combinations are being considered?

A complete matrix of the HR1P and HR2P peptides from MERS-CoV, SARS-CoV, HCoV-229E, HCoV-NL63, and HCoV-OC43.

What are the main hypotheses being tested?

Based on their previous MERS-CoV study, they hypothesize that HR1- and HR2-derived peptides could also inhibit other coronaviruses in vitro.
Since the OC43-HR2P-derived peptide EK1 showed the best inhibitory effect on the five human coronaviruses they evaluated in vitro, they hypothesize that this would be a potential pan-coronavirus therapeutic in vivo.

Study population:

What is the model system (e.g., human study, animal model, cell line study)?

EK1 and peptide homology models were derived from Swiss-Model. They were further optimized by molecular dynamics simulations.
Pseudoviruses encoding (1) the respective S proteins, (2) the respective VSV-G proteins, or (3) a defective luciferase-expressing HIV-1 genome from various viruses were created (i.e., from MERS-CoV, SARS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, Rs3367 SL-CoV, WIV1 SL-CoV, SHC014 SL-CoV). Then, the viral replication was tested on Huh-7 cells (ACE2/293 T for SARS-CoV) in the absence or presence of respective viral-derived peptides besides EK1 in original and scrambled versions as control.
The fluorescence model measured cell-cell fusion from transfected 293 T effector cells to Huh-7 target cells in the absence or presence of the respective viral-derived peptides besides EK1 in original and scrambled versions as control.
An FCM model measured virion-cell fusion of SARS-CoV and MERS-CoV Blam-Vpr to Huh-7 and ACE2/293 T cells in the absence or presence of EK1.
The inhibition of the respective viral-derived peptides or EK1 on viral replication in different cell lines was verified by Kärber TCID50 viral titer based on CPE 5 days after infection (i.e., HCoV-OC43 in HCT-8, HCoV-229E in A549, HCoV-NL63 in LLC-MK2).
For MERS-CoV-induced CPE quantification, they used a Calu-3 cell culture in a modified assay. In the absence or presence of the respective peptide or EK1, MOIs and CPE were observed 24 hours to 72 hours after infection, and CPE was then quantified in a Vero E6-based assay 72 hours after the infection.
A mouse model was used to verify the prophylactic and therapeutic effect of EK1 on MERS-CoV and HCoV-OC43 infection.
In vivo fluorescence imaging of the peptides was performed on mice as well.
The cytotoxicity of the peptides was measured after a two-day incubation of 293 T, ACE2/293 T, Huh-7, A549, LLC-MK2, and Calu-3 cells by cell counting.
Bodyweight, ALT, and creatinine of the mice were measured as indices for in vivo safety.

What is the sample size? If multiple groups are considered, give sample size for each group (including controls).

5 8-weeks-old female Balb/c mice in the low-dose EK1 safety group
5 8-weeks-old female Balb/c mice in the high-dose EK1 safety group
5 8-weeks-old female Balb/c mice in the PBS-controlled safety group
11 newborn Balb/c mice in the EK1 prevention HCoV-OC43 group
11 newborn Balb/c mice in the EK1 treatment HCoV-OC43 group
11 newborn Balb/c mice in the untreated HCoV-OC43 control group
11 newborn Balb/c mice in the untreated base control group
6 DPP4 mice in the EK1 prevention MERS-CoV group
6 DPP4 mice in the EK1 treatment MERS-CoV group
6 DPP4 mice in the untreated MERS-CoV group
3 Balb/c mice for EK1 fluorescence imaging
3 Balb/c mice for untreated fluorescence imaging control
Triplicate sample measurements are used where applicable

Treatment assignment:

Is the study randomized?

Randomized selection of mice.

Provide other relevant details about the design.

Only one animal line is used within each subgroup. It seems unclear though if the offsprings are randomized after birth. In the MERS-CoV subgroup, the authors didn't include an untreated base control though.

Outcome Assessment:

Describe the outcome that is assessed and whether it is appropriate.

For example: Is the outcome assessed by a clinician or is it self-reported? Is the outcome based on viral load or a functional measurement (e.g., respiratory function, discharge from hospital)? What method is used to measure the outcome? How long after a treatment is the outcome measured?

Are outcome measurements complete?

They are complete.

Are outcome measurements subject to various kinds of bias?

Different cell lines, mouse lines, and assays are used. But all the subgroups include separate controls.

Statistical Methods Assessment:

What methods are used for inference?

TCID50 is used where applicable.

Are the methods appropriate for the study?

TBA

Are adjustments made for possible confounders?

The MERS-CoV Calu-3 assay is a modified version based on one of their previous papers.

Results Summary:

What is the estimated association?

The survivability of the mice is measured absolutely after the 2 weeks.

What measures of confidence or statistical significance are provided?

Depicted charts use means and include standard deviations from triplicate samples. P values for significant charts are provided.

Interpretation of results for study population:

Can we make a causal interpretation for the individuals in the study of drug -> outcome, such as "taking drug A improves the likelihood of survival twofold over taking drug B."

In all groups, prophylactically EK1-treated mice showed 100% survivability within two weeks. In all groups, untreated mice died within 7 to 10 days after infection. The survivability of EK1-treated mice was 66.7% for HCoV-OC43 and 75% for MERS-CoV. No causal interpretation is possible for human SARS-CoV-2 infections. EK1 might have an inhibitory effect on the SARS-CoV-2 S2 region of its spike protein.

Are there identified side effects or interactions with other drugs?

In vitro, concentrations up to 200 times the inhibitory IC50 HCoV cell-cell fusion dosage were tested without any increase in cell toxicity in all the cell lines they used. In vivo, in healthy mice, no dose-dependent antibody increase could be detected 2 weeks after EK1 administration (i.e. for 20 and 100mg/kg). Bodyweight didn't change within these 2 weeks either. ALT and creatinine levels showed no significant difference to PBS-treated mice within 5 days. EK1 can enrich primarily in the liver and the kidneys but probes of these organs didn't show any histopathological damage after 4 weeks.

Are there specific subgroups with different findings?

No different baselines were measured.

Extrapolation of conclusions to other groups of individuals not specifically included in the study:

If the study is an animal study, which animal and how relevant is that model?

The model seems to be appropriate to initially determine effectiveness and safety. Aged Balb/c mice showed greater severity though and might have been preferred over newborn ones [10.1016%2Fj.coviro.2015.06.009].

Summary of reliability

Although the authors evaluate not only SARS-CoV but also SL-CoV S protein-mediated cell-cell fusion, the data is not separately shown. The mechanism on the S protein has not been verified by microscopy. The study shows a clear inhibition effect on viral replication and cell survivability but it doesn't show that EK1 is a sole S protein inhibitor even though it is derived from the S protein. No controls have been used for other viral proteins; neither in vitro nor in their simulations. Given their data, it still seems possible to me that EK1 also affects other proteins of coronaviruses. This question became relevant only as of lately.

Progress

Check off the components as they are completed. If the component is not applicable, check the box as well.

[x] 1-2 sentences introducing the study and its main findings
[x] Describe How many/what drugs/combinations are being considered
[x] Describe the model system
[x] What is the sample size?
[x] What countries/regions are considered
[x] What is the age range, gender, other relevant characteristics
[x] Describe study setting
[x] Describe other specific inclusion-exclusion criteria
[x] Describe how treatments are assigned
[x] Describe randomization (or not) and other relavent details about the design
[x] Describe the outcome that is assessed and whether it is appropriate.
[x] Describe whether the outcome measurements are complete
[x] Are outcome measurements subject to various kinds of bias?
[ ] Describe methods used for inference
[ ] Describe whether the methods are appropriate for the study
[ ] Are adjustments made for possible confounders?
[ ] Describe the estimated association
[x] What measures of confidence or statistical significance are provided?
[x] Describe whether a causal interpretation can be made
[x] Are there identified side effects or interactions with other drugs?
[x] Are there specific subgroups with different findings?
[x] If the study is an animal study, which animal and how relevant is that model?
[x] If it is a human study, what characteristics of the study population may support/limit extrapolation?
[x] Summary of reliability

Feedback regarding the following questions would be particularly welcome:

[ ] They provide raw data that could be statistically verified with their figures.
[ ] Is their homology model accurate and complete?
[ ] Are their in vitro and mouse models appropriate?
[ ] Do you agree with my interpretation regarding the S protein? Can this drug be called a sole S protein inhibitor? Does the homology model prove that EK1 only affects the S protein?

greenelab / covid19-review