Using in vivo ARDS models to screen for potential COVID-19 treatments

Solutions to the current COVID-19 pandemic involve preventing coronavirus infection through vaccination and treating the severe clinical symptoms of the disease, including acute respiratory distress syndrome (ARDS). Establishing in vitro screening for potential treatments, though, can be challenging, due to the limited number of appropriate research models available, and the need to perform preclinical testing in a biosafety level 3 facility. These challenges add cost and complexity to the development of a coroanvirus treatment.

Disease models of ARDS (those that are independent of SARS viruses); however, are well-established for preclinical testing of respiratory drugs. We recently explored the use of preclinical ARDS models for testing a potential COVID-19 drug candidate and want to share some results.

Relationship Between ARDS & COVID-19

COVID-19 is a disease caused by strain SARS-CoV-2, which is a novel member of the coronavirus family. The genome of SARS-Cov-2 is 96% identical to a coronavirus found in bats and 79.6% identical in sequence to SARS-Cov, the pathogen of the severe acute respiratory syndrome (SARS), which caused two outbreaks, both originating in China but spreading worldwide between 2002 and 2004.

COVID-19 affects different people in different ways and to different extents. The majority of those infected with COVID-19 experience only mild symptoms, however some go on to develop serious, life-threatening symptoms.

Approximately one in six COVID-19 patients have difficulty breathing, and about 40% of these patients then develop acute respiratory distress syndrome (ARDS). Estimated mortality rate for patients with severe COVID-19-related ARDS is 80%.

Estimated mortality rate for patients with severe COVID-19-related ARDS is 80%

The underlying clinical pathology of ARDS relates to a severe immune response causing cytokine release and hyper-inflammation in the lungs that affects the upper and lower airways. In the clinic, increased serum levels of inflammatory cytokines IL-6, IL-1beta and TNFα have been associated with disease severity and death in COVID-19 patients.

Identifying potential treatments for coronavirus-triggered ARDS is therefore a key priority.

The challenge is how to do this rapidly and efficiently.

Types of Models Needed to Screen Potential COVID-19 Treatments

Preclinical models of disease are useful tools for mimicking human disease; however, they mimic only certain aspects of the disease. When choosing a preclinical model to test the efficacy of a compound, you need to consider which aspect of the human disease you are targeting. For example, if your compound inhibits IL-6, then it is important your preclinical model induces increased levels of IL-6.

SARS-CoV-2 is a viral pathogen, which affects the respiratory tract, initially infecting the cells of the upper respiratory tract, but in more severe cases spreading to the lower respiratory tract and causing pneumonia and ARDS. In these more severe cases, ARDS is characterized by a low blood oxygen level, difficulty in breathing and respiratory failure. This respiratory failure is the leading cause of death in COVID-19 patients.

For the development of therapeutics that target the effects of inflammation you need a preclinical model that reflects COVID-19 pathophysiology; including pathology, cytokine release and airway cell inflammation, but the model does not necessarily need to mimic the actual pathogen insult.

Species selection

Although COVID-19 is a zoonotic virus, it is relatively species-specific and standard inbred mice are not an appropriate vector because they readily clear the virus from the respiratory tract and are not infected.

It has been shown that COVID-19 can cause infections in bats, cats and NHPs, although none of these are appropriate species to test large numbers of novel compounds. Ferrets are another potential model species that have been successfully used for studying influenza viral infections; however, for COVID-19 infections, ferrets show only a limited pathology with the majority of the infection remaining in the upper respiratory tract.

Selecting the most appropriate species for testing potential COVID-19 treatments is crucial to the accuracy of the drug development process; however, deciding which species is the most appropriate is not straight forward and should always take into consideration the 3Rs principles: replacing animal model with non-animal alternative methods when they exist, reducing the number of animals used and refining the procedures from animal welfare perspective.

So, what are your options? Which available and reliable preclinical models are appropriate for screening COVID-19 potential treatments?

Viral Model Screening Challenges

Several strategies can be used to develop models of human viral infections in mice. One is to over-power the mouse’s immune system with an extremely high titre of virus. This method has worked for viruses such as rhinovirus and Herpes simplex virus, but these models don’t always display the same pathology as those seen in humans. Another strategy is to adapt the viral strain to the animal model, so it can invade and infect cells in the same way as it does humans.

Alternatively, and the approach being used to model COVID-19 infection, is to use a humanized transgenic mouse strain. This is the best model to assess the efficacy of viral vaccines and compounds that target the virus’ ability to bind to hosts cells.

However some practical challenges exist for using transgenic mice as models for SARS-CoV-2:

Access to a BSL-3 facility: This is a facility that houses operations involving potentially lethal or ‘exotic’ diseases that are contracted via inhalation. These facilities are not widely available commercially, as many are governmental-owned or based in universities. Learn more about BSL labs on the CDC website
Limited supply of human transgenic mice: The K18-hACE2 transgenic mouse (which expresses the human ACE2 receptor used by SARS-CoV-2 to gain entry to cells) was developed in 2007, but availability of this transgenic model is currently very limited with long delivery wait times.

Given these limitations, viral models may be limited options for efficacy studies.

So, what about using an ARDS-specific model instead of a COVID-19 model to mimic advanced disease for testing treatment options?

Alternate Strategy: Use In Vivo ARDS Models

To test compounds or treatments that may help to prevent ARDS and downstream hyper-inflammation/cytokine storm in the lungs of COVID-19 patients, a more general in vivo model of ARDS may be appropriate.

Many well-validated and well-studied models of lung injury and ARDS are available in rodents in which an inflammatory agent is administered and the associated pathology measured.

What is an ARDS model?

The leading cause of ARDS in humans is lung infection. This can be modeled in mice via two methods, either by using a non-infectious inflammatory substance, such as lipopolysaccharide (LPS) or by using a live pathogen, such as influenza.

By administering these insults to the lung, clinically relevant endpoints–including injury to the epithelial cells and an acute inflammatory response in the air spaces (inflammatory cell recruitment)–can be initiated.

Lipopolysaccharide (LPS)-Induced Model of Pulmonary Inflammation

One of the simplest models of ARDS that can mimic COVID-19 inflammation is the LPS-induced model of pulmonary inflammation, a reliable, rapid mechanistic model.

LPS is a sugar, purified from the cell wall of gram-negative bacteria. When it is delivered via aerosol to the lungs of rodents, it induces a Th1 immune response, causing an influx of inflammatory cells (predominantly neutrophils) into the airways as well as causing a large increase in levels of cytokines in the lung.

Although this doesn’t mimic the full pathology induced by COVID-19, it does mimic the Th1 inflammation observed in COVID-19 patients. This model has been validated in several animal species including rodents.

Figure 1 below shows recruitment of inflammatory cells and release of cytokines induced by the administration of LPS in mice.

Administration of the steroid dexamethasone, the PDE4 inhibitor roflumilast and p38 inhibitors (PH-797804 and SB203580) results in a significantly significant reduction in LPS-induced lung inflammation.

Viral Influenza (H1N1) Mouse Model

Another potentially more appropriate model of ARDS is to use a live pathogen to induce ARDS symptoms. Many characterized models of infection exist that use both bacteria and viruses; however, as SARS-CoV-2 is a viral respiratory pathogen, a model of ARDS, induced by another respiratory virus, such as influenza, could be an appropriate model to use.

Although influenza and COVID-19 are different viruses, they both infect the respiratory tract and induce potentially fatal inflammation. Aspects of the clinical pathologies of the viruses do differ, but they also have many similarities. Both viruses in the clinic (and in preclinical models) can cause severe pneumonia, respiratory distress and death.

Unlike SARS-CoV-2, an administration of a relatively low titre of H1N1 intranasally to an inbred strain of mouse can induce a lower respiratory tract infection, which reduces lung function, causes influxes of inflammatory cells, including neutrophils and cytokines (IL-6, IL-1β and IFNγ), and, at higher titres, could cause mortality.

Figure 2 shows the recruitment of neutrophils and the release of cytokines during the week following the administration of increasing titres of H1N1 in mice.

In addition to observing an increase in inflammatory cells (neutrophils), we noted that animals also showed clinical signs of infection, such as hunched posture, piloerection and weight loss. The severity of these clinical signs, correlated with the titre of H1N1 administered: the higher the titre, the more severe the clinical signs. Animals being administered 400 pfu H1N1 were euthanased on Day 6 according to pre-established humane end points.

Lung pathology images from Day 7 are shown in Figure 3 below from mice who had been administered with 40 pfu of H1N1. These images show signs of very poor lung function, including destruction of alveoli, marked inflammation of the lung interstitium and alveoli, and a hyaline-like membrane formation.

Read the poster from the study.

Conclusion

By using a viral-induced model of ARDS, efficacy of novel treatments of respiratory inflammation and generic antivirals can be assessed without requiring a BSL-3 facility.

The model of H1N1 viral infection is well-established and can be used to assess levels of inflammation, including but not limited to inflammatory cells, cytokines, lung function (both invasive and non-invasive) and pathology. These models take less than 2 weeks to run and, depending on the type of endpoints being analysed, can yield data within those 2 weeks.

Targeting respiratory inflammation is a viable treatment option for COVID-19. By being able to better prevent, reverse or reduce the severity of the cytokine storms and hyper-inflammation induced by respiratory viruses, such as COVID-19, the number of fatalities caused by such severe diseases could be drastically reduced.

Note: Animal care and use was performed in conformance with the Guide for the Care and Use of Laboratory Animals in an AAALAC-accredited facility. Humane endpoints were observed and studies were approved by the Animal Welfare Ethical Review

Using acute respiratory disease syndrome (ARDS) in vivo models to screen for coronavirus inflammation treatment

Relationship Between ARDS & COVID-19