Interesting Preprints/Papers on Coronavirus

#21

Mechanical Ventilator Milano (MVM): A Novel Mechanical Ventilator Designed for Mass Scale Production in Response to the COVID-19 Pandemics

This is the first paper for ventilator design that I’ve seen on arxiv, so seemed useful for a quick review. The goal of the work is to create a design for a simple electro-mechanical ventilator that can be built with off-the-shelf hardware components. It’s not a certified medical device though.

The background section explains that there are two types of ventilation. Positive end-expiratory pressure (PEEP), where the pressure in the lungs is maintained above atmospheric pressure at the end of the respiration cycle. The other method is pressure support ventilation (PSV), where the patient initiates each breath, and the ventilator provides support with preset pressure value.

The current design pulls from the old Manley ventilator, with the difference that this design uses electrically driven pneumatic valves. This current design follows guidelines for “Rapidly Manufactured Ventilator Systems” recently released by the UK medicine&healthcare products regulatory agency. The MVM delivers PEEP pressure to patients in the range 20-30 mbarg (1 mbarg is atmospheric pressure plus pressure given by \textrm{H}_2\textrm{O} column 1cm in height.

The MVM. consists of a small number of components: a medical care flowmeter, an oxygen therapy humidifier, a non-vented non invasive ventilation mask. The parts have been selected so that the total cost only amounts to a few hundred euros.

#22

UV Sterilization of Personal Protective Equipment with Idle Laboratory Biosafety Cabinets During the COVID-19 Pandemic

This is a preprint that will shortly be posted on medrxiv. One of the major issues that’s been facing medical facilities has been PPE (personal protective equipment such as N95 masks, or even surgical masks). This paper notes that a number of hospitals have already been using UV light to sterilize N95 masks, but notes that most hospitals don’t have the space or equipment. The preprint suggests using biosafety cabinets (BSCs) for this purpose. These are common in many academic centers and teaching hospitals already. The main impediment apparently is the possibility that UV levels vary within BSCs.

The authors tested their procedure in two randomly chosen idling BSCs in their facility and observed a maximum ratio between max/min recorded intensities to be 1.98. This leads them to conclude that a N95 mask placed in a BSC with fluence of 100 \mu\textrm{W}\textrm{cm}^{-2} to be sanitized after 15/20 minutes for each side.

The authors measured intensities in the BSCs they ran experiments in, used estimates from the literature for the time needed to deactivate viral ssRNA, then did a simple calculation to estimate the time needed. They note their method needs to be tested with larger experiments and virology work, but they estimate that in an ideal setting, nearly 121 million masks/month can be sanitized for re-use under ideal conditions. They also provide a simple sanitization protocol.

The authors note that one limitation is that N95 masks have multiple layers, and it’s possible viral droplets could penetrate into the deeper layers which UV santiziation couldn’t help with. Another concern is that UV sanitization could damage the polymers in the N95 mask

#23

Is There a Role for Tissue Plasminogen Activator (tPA) as a Novel Treatment for Refractory COVID-19 Associated Acute Respiratory Distress Syndrome (ARDS)?

There’s a MIT press release about this paper as well.

A big cause of the deaths from the coronavirus is due to ARDS (acute respiratory distress syndrome). Past work has noted that ARDS causes deposition of fibrin (protein involved in blood clotting along with platelets) in airspaces and lung parenchyma along with fibrin platelet microthrombi deposited in the pulmonary vasculature. In other words, blood clots are deposited into the lungs, causing damage.

tPA is a protein involved in breaking down blood clots. The protein can be manufactured using recombinant techniques and is widely used as a treatment for strokes (where blood clots obstruct blood flow to the brain). Given the current drastic shortage of ventilators with the ongoing coronavirus crisis, the authors posit that using tPA as a stopgap treatment for ARDS may have useful effect. The use of agents such as tPA is typically considered higher risk, but may be necessary given the current crisis.

The paper suggests that using experience from strokes, intravenous administration of tPA may be useful. They suggest that unlike strokes, where a brief dose is used, for coronanavirus, a better treatment may be to have a first dose of 25 mg tPA administered over 2 hours, followed by another 25 mg tPA dose over the subsequent 22 hours. They suggest patients with severe ARDS where ventilators are not available may be good candidates.

#24

Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma

In this early study, the treatment of 5 critically ill patients with COVID-19 and ARDS, administration of convalescent plasma from recovered patients containing neutralizing antibodies was followed by an improvement in clinical status. The donors were patients between 18 and 60. Patients were between 36 and 73, with 2 women. The following criteria were used to select patients:

  • severe pneumonia with rapid progression and continuously high viral load (despite antiviral treatment)
  • \textrm{P}_{\textrm{AO}_2}/\textrm{F}_{\textrm{IO}_2} < 300
  • had been on mechanical ventilation.

Patients received transfusion with convalescent plasma with a SARS-CoV-2-specific antibody (IgG) binding titer greater than 1:1000 and a neutralization titer greater than 40. Plasma was administered 10-22 days after admission.

Changes in body temperature, Sequential Organ Failure Assesment (SOFA), \textrm{P}_{\textrm{AO}_2}/\textrm{F}_{\textrm{IO}_2}, viral load, serum antibody titer, routine blood biochemical index, ARDS, and ventilator and ECMO supports.

After treatment, body temperatures stabilized in 3 days for 4 out of 5 patients. Viral loads also decreased or became negative in 12 days.

The authors note that this is a small study with no controls, and it is unclear whether patients would have improved without transfusion of convalescent plasma.

#25

Efficacy of hydroxychloroquine in patients with COVID-19: results of a randomized clinical trial

There’s some really nice commentary about this trial on Derek Lowe’s blog. This paper discusses results of a small trial using hydroxychloroquine on 62 patients (31 treatment, 31 control). This trial is actually blinded, randomized, and controlled unlike earlier trials.

#26

A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2

This work used the Immune Epitope Database and Analysis Resource (IEDB) and the Virus Pathogen Resource (ViPR) to catalog immune response data for other coronaviruses such as SARS-CoV. The authors identify multiple regions in SARS-CoV that have high sequence homology to SARS-CoV-2. SARS-CoV has well characterized epitope responses. Here’s a nice graphical abstract.

image

The main challenge the authors faced is that right now, not much is known about which SARS-CoV-2 sequences are recognized by human immune responses. This work uses known epitope information for SARS-CoV, and MERS to compile known epitope regions for these other coronaviruses, then try to map these sequences back onto the SARS-CoV-2 sequence. As a parallel line of analysis, the authors use Bepipred 2.0 and Discotope 2.0 (machine learning based methods) to predict B-cell epitopes from the SARS-CoV-2 sequence. To predict T-cell epitopes, the authors use TepiTool.

The authors found a number of overlaps between the epitopes identified by the two approaches.

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#27

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven
Protease Inhibitor

This paper is a little old, but there was just released an interesting sciencemag article discussing how these compounds are moving into the clinic already! So it seems worthwhile to go back and read the paper that inspired this approach.

The paper shows that SARS-CoV-2 infection depends on human host factors ACE2 and serine protease TMPRSS2 and that this can be blocked by a clinically proven protease inhibitor. Vesicular stomatitis virus (VSV) particles (which are replication defective) are useful model system that faithfully reflect coronavirus host cell entry. The paper studies which human cell lines are are susceptible to entry by SARS-CoV-2 using VSV as a proxy system. All cell lines tested were susceptible to entry. Cell lines showed a similar spectrum for cells lines as SARS-CoV, suggesting that a similar use of entry cell receptors. In particular, ACE2 appears to be used by both.

SARS-CoV uses the endosomal cystein proteases cathepsin B/L and serine protease TMPRSS2 for S protein priming, and inhibiting both is useful for robustly blocking cell entry. Serine protease inhibitor camostat mesylate was found to partially block SARS-CoV-2 entry into cell lines.

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#28

The FDA-approved Drug Ivermectin inhibits the replication of SARS-CoV-2 in vitro

There’s a nice twitter thread that provides some analysis of this paper.

Ivermectin is an anti-parasitic drug that’s widely available and hows reduction of the virus in a Vero cell culture model. Past work has shown that ivermectin can limit RNA virus infection for other viruses such as West Nile Virus.

To test the antiviral activity of ivermectin, authors infected Vero cells with SARS-CoV-2 followed by the addition of 5 \mu\textrm{M} ivermectin

#29

A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV

There’s a nice write-up press release about this article from Scripps.

This paper determines the crystal structure of CR3022, a SARS-CoV neutralizing antibody, in complex with the receptor binding domain of the SARS-CoV-2 S spike protein. CR3022 targets an epitope that’s highly conserved between SARS-CoV and SARS-CoV-2. This site is distal (distant) from the receptor binding site

image

Out of 28 residues in the epitope, 86% are conserved between SARS-CoV and SARS-CoV-2. However, binding is much tighter to SARS-CoV (1 nM) than SARS-CoV-2 (115 nM). The epitope of CR3022 doesn’t overlap with the ACE2 binding site.

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#30

Anti–spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection

There’s a nice twitter thread discussing this paper.

This paper is a little older, from last year, focusing on SARS-CoV. Macaque monkeys were infected with SARS-CoV. Anti-spike IgG antibodies caused acute lung injury (ALI) by skewing inflammation responses. Macaque monkeys injected with antibodies then infected with SARS-CoV developed a worse form of the disease. The mechanism for this appears to be that the antibody binds to the virus, facilitating uptake by macrophages, leading to macrophage stimulation and production of proinflammatory cytokines. As the twitter thread points out, this could mean that targeting the spike protein as the vaccine antigen could have detrimental effects, suggesting that SARS-CoV-2 vaccines may need to target nucleocapsid or other antigens.

#31

No evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe COVID-19 infection.

This is a really interesting recent paper that makes the situation around Hydroxychloroquine even murkier. This was a small trial with 11 patients who received hydroxychloroquine (600 mg/d for 10 days) and azithromycin (500 mg Day 1, 250 mg Days 2-5) at the same doses as the Gautret et al. paper. There were 7 men and 4 women patients, 8 of whom had significant comorbidities. 10/11 patients had fevers when treatment started. In 5 days, one patient died and 2 were transferred to the ICU. In one patient, treatment was discontinued in 4 days because of a prolongation of the [QT interval] was observed under treatment. Nasal swabs in 10/11 patients (excluding patient who died) were still positive for 8/10 patients on days 5/6 after treatment initiation.

The authors conclude that treatment with hydroxycholoroquine/azithromycin doesn’t show benefit of clinical benefit. My two cents is that it’ll be interesting larger controlled trials to come out. It’s clear hydroxycholoroquine+azithromycin isn’t a magic treatment, but perhaps it has some effect over baseline. However, this possibility grows dimmer as more studies with mixed results come out. We’ll have to wait and see…

#32

Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications

There’s some analysis on Derek Lowe’s blog of this paper.

Plasma was recovered from 175 Covid19 recovered patients with mild symptoms and screened for spike binding antibody. There were 10 patients who had no detectable level of antibodies! Younger patients had lower levels of antibodies than older patients as well. This paper suggests that immunity to the disease post infection may be a complex thing, since some patients who had the disease may actually not have immunity

#33

Structural and functional analysis of a potent sarbecovirus neutralizing antibody

This work identifies multiple monoclonal antibodies for the SARS-CoV-2 S protein identified from memory B cells of a SARS survivor infected in 2003. One of these antibodies, S309, is a potent neutralizer of both SARS-CoV and SARS-CoV-2. The paper uses Cryo-EM show that the antibody recognizes a glycan containing epitope and doesn’t compete with receptor attachment. As a quick note about terminology here, both SARS-CoV and SARS-CoV-2 belong to the sarbecovirus subgenus.

This work may be useful for the generation of antibody therapeutics. The paper notes that passive administration of antibodies could provide immediate protection and serve as a complement to prophylactic vaccine design efforts.

#34

SARS-CoV-2 infects T lymphocytes through its spike protein-mediated membrane fusion

Some studies have found that lymphocytopenia might be connected to mortality for covid patients. Similar observations have been made for MERS patients as well. This suggests that SARS-CoV-2 might be able to infect T-cells. This work set out to investigate this hypothesis.

SARS-CoV-2 and SARS-CoV were packaged as pseudoviruses. These pseudoviruses were used to infect two lymphocyte T-cell lines (MT-2 and A3.01) with very low expression of hACE2 mRNA. This suggests that the spike S protein might mediate infection on cells expressing low hACE2, possibly explaining why the transmission rate is so high. CD147 is a possible entry route into T-cells.

Experiments with a peptide known to block receptor mediated infection show that SARS-CoV-2 entry does seem to be receptor mediated, since the peptide inhibits T-cell infection.

Experiments were also performed with live virus showing that live SARS-CoV-2 can infect MT-2. Interesting, SARS-CoV-2 can infect T-cells but does not seem to replicate in them. Similar behavior has been observed for MERS.

#35

COVID MOONSHOT: Docking and free energy calculations

This is a set of slides detailing work by the Folding@Home Covid-moonshot. There are some excellent scripts in the Github repo (https://github.com/foldingathome/covid-moonshot). Unfortunately a lot of these scripts seem to depend on openeye, so there might not be a good way to replicate with open source tools.

The slides discuss how since there were many diverse scaffolds submitted, accurate scoring became tough. Compound synthesis for testing was prioritized with alchemical free energy calculations. Almost 6000 free energy calculations are being run on Folding@Home. These calculations use the new Parsley open force field for small molecules.

#36

The QT Interval in Patients with SARS-CoV-2 Infection Treated with
Hydroxychloroquine/Azithromycin

The QT interval is an ECG measurement used to measure properties of the heart. An abnormally long QT interval is associated with higher risk of arrhythmia and sudden cardiac death. Which makes the results of this paper a little distressing.

QT interval was retrospectively tracked in 84 adult patients treated with hydroxycholoroquine/azithromycin combo. QT interval prolonged maximally from baseline in days 3-4. In 30% of patients it increased by over 40ms. In 11%, it increased over 500ms, representing a high risk group for arrhythmia. Given how widely the hydroxychloroquine/azithromycin combo is used, this is a very worrying statistic, suggesting that QT interval should be tracked closely during the course of treatment.

#37

Compassionate Use of Remdesivir for Patients with Severe Covid-19

There’s a nice twitter thread discussing this article.

Remdesivir was provided on a compassionate use to patients with covid-19. Patients had confirmed covid-19 and oxygen saturation of 94% or less while they breathed ambient air or were on oxygen support. Patients received 10 day course of remdesivir, with 200 mg intravenously on day 1, then 100mg daily for remaining 9 days.

61 patients received remdesivir. 8 patients’ data could not be analyzed due to various reasons. Of 53 who could be analyzed. At baseline, 57% of patients (30 patients) were on mechanical ventilation and 8% (4 patients) on ECMO. The median follow-up was 18 days. 68% of patients had an improvement in oxygen support class. 17 of 30 intubated patients were extubated. 25 patients (47%) were discharged. 7 patients (13%) had died.

Note that this study was open-label and that there was no control group. For a comparison, the paper notes that a recent randomized-controlled trial of lopinavir-ritonavir had 28-day mortality of 22%. However only 1 of 199 patients where intubated at the start of that trial.

#38

There is an interesting approach to dealing with COVID-19 using genetic modifying technology. This paper https://www.biorxiv.org/content/10.1101/2020.03.13.991307v1.full.pdf describes the use of a CRISPR system that can be given guide-RNA sequences that allow the CRISPR enzyme to bind to the SARS-CoV-2 viral RNA and cut it into pieces. The method is called PAC-MAN for (ProphylacticAntiviral CRISPR in huMAN cells). One of the big challenges is finding a way to deliver it into the infected cells. One proposal is to take a SARS-CoV-2 virus and replace its viral RNA with an mRNA that can be translated into CRISPR-cas13 enzyme and a guide RNA for finding the SARS-CoV-2 viral RNA.

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#39

News Feature: Avoiding pitfalls in the pursuit of a COVID-19 vaccine

This is an informative news article that talks through some of the pitfalls facing vaccine development inlcuding Antibody Dependent Enhancement and Th2 immunopathology causing allergic inflammation. Some researchers believe Th2 immunopathology is understudied as a potential risk factor for coronavirus vaccine development since previous studies with SARS vaccines showed risk of Th2 issues.

#40

SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness

This is a preprint from Moderna detailing the design of their mRNA vaccine candidate and some results from mouse models showing the candidate induces neutralizing antibody and CD8 T cell responess. Some protective effect is also shown in lungs and noses of mice against infection with covid.