IN VITRO AND ANIMAL MODELS FOR SARS-COV-2 RESEARCH

              IN VITRO AND ANIMAL MODELS FOR SARS-COV-2 RESEARCH

 

I.             Introduction

A coronavirus is a positive sense, single-strand enveloped RNA virus belonging to the family Coronaviridae.  Coronavirus disease 2019 (COVID-19) is a respiratory infection that has infected more than 150 million people and led to more than 3.1 million deaths worldwide since it was first discovered in late 2019 in Wuhan, China.  The etiological agent of COVID-19 has been identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), one of a large family of viruses that usually cause mild to moderate upper-respiratory tract illnesses, such as the common cold, but which have included three particularly serious – and deadly – strains over the past two decades.  

This essay summarizes an article by Kazuo Takayama titled “In Vitro and Animal Models for SARS-CoV-2 research,” published on May 30, 2020, in Trends in Pharmacological Sciences (https://www.cell.com/trends/pharmacological-sciences/fulltext/S0165-6147(20)30129-2), describing the research methods being employed at that time to achieve the efficient development of therapeutic agents for COVID-19.  The article explains that for the research to be effective, it would have to employ models that can faithfully reproduce the behavior of the virus and the pathology of COVID-19 in humans.  The article reviews relevant cell lines, organoids, and animal models relevant to that research.

At the time the article was published, an estimated 5.4 million people around the world had been infected with COVID-19, of whom an estimated 343,000 had died. Today, those figures are 150 million and nearly 3.2 million, respectively.

I.             Cell Lines and Organoids for SARS-CoV-2 Research

The Takayama article argues that an in vitrocell model for SARS-CoV-2 research is essential for understanding the viral life cycle, for amplifying and isolating the virus for further research, and for preclinical evaluation of therapeutic molecules.  The article reviews the cell lines used to replicate and isolate SARS-CoV-2 as well as organoids that could be used to examine the effects of SARS-CoV-2 infection on specific human tissues.

A.   Cell Lines 

In humans, airway epithelial cells highly express the putative SARS-CoV-2 entry receptor known as angiotensin-converting enzyme 2 (ACE2) as well as transmembrane serine proteinase 2 (TMPRSS2), the receptor that the virus uses to prime the S protein (the spike protein of SARS-CoV-2).  SARS-CoV-2 infection experiments using primary human airway epithelial cells have been found to have cytopathic effects 96 hours after the infection.  However, primary human airway epithelial cells are expensive and do not proliferate indefinitely.  Instead, researchers have used infinitely proliferating cell lines in SARS-CoV-2 infection experiments.  These include Caco-2, Calu-3, HEK293T, and Huh7.  The drawback of these cell lines is that they do not accurately mimic human physiological conditions and they generate low titer of infectious SARS-CoV-2. Despite these limitations, studies using these cell lines can yield important lessons about virus infection and replication.

For efficient SARS-CoV-2 research, however, a cell line that can easily replicate and isolate the virus is necessary.  Vero cells, which have given high titer of viral particles, were isolated from the kidney epithelial cells of an African green monkey in 1963 and have been shown to not produce interferon (IFN) when infected with Newcastle disease virus, rubella virus, and other viruses.  This IFN deficiency allows SARS-CoV-2 to replicate in Vero cells.  Of the Vero cell clones, Vero E6 is the one most frequently used to replicate and isolate SARS-CoV-2 because the cells highly express ACE2 on the apical membrane domain. However, the expression level of TMPRSS2 is low in this clone.  TMPRSS2-overexpressing Vero E6 cells have therefore been used to enhance the replication and isolation efficiencies of SARS-CoV-2 in Vero E6 cells.  Research has found that the viral RNA copies in the culture supernatants of these cells are more than 100 times higher than those of Vero E6 cells, suggesting that it would be possible to isolate higher titer virus using TMPRSS2-overexpressing Vero E6 cells.

B.   Organoids

Organoids are composed of multiple cell types and model the physiological conditions of human organs.  Because organoids have the ability to self-replicate, they are also suitable models for large-scale screening in drug discovery and disease research.  In addition to the lung damage caused by pneumonia, SARS-CoV-2 affects the kidney, liver, and cardiovascular system, among other systems.  Researchers have generated bronchial organoids and human lung organoids for SARS-CoV-2 research.  These organoids are permissive to the SARS-CoV-2 infection and allow researchers to evaluate antiviral effects of COVID-19 candidate therapeutic compounds, including camostat.  

Other research has shown that the supernatant of SARS-CoV-2-infected kidney organoids differentiated from human embryonic stem cells can efficiently infect Vero E6 cells, showing that the kidney organoids produce infectious progeny virus.  Still other research has shown that human liver ductal organoids are permissive to SARS-CoV-2 infection and support replication. In one interesting finding, virus infection impaired the bile acid-transporting functions of cholangiocytes, which might help explain the bile acid accumulation and consequent liver damage in patients with COVID-19.  

The intestine is expected to be another viral target organ.  It has been reported that human intestinal organoids established from primary gut epithelial stem cells support SARS-CoV-2 replication.  It has also been shown that SARS-CoV-2 can directly infect human blood vessel organoids differentiated from human induced pluripotent stem cells.  Researchers confirmed the presence of viral elements within endothelial cells and an accumulation of inflammatory cells. The studies suggest that SARS-CoV-2 infection induces endotheliitis in several organs as a direct consequence of virus involvement.  While organoids can reproduce the pathology of COVID-19 in specific tissues on which they are modeled, they cannot reproduce the systemic symptoms associated with whole body responses to the viral infection.

II.           Animal Models for SARS-CoV-2 Research

The Takayama article posits that the complex pathophysiology of COVID-19 cannot be understood unless tissue-specific and systemic virus-host interactions can faithfully be replicated.  While cell lines and organoids provide a faster means of studying the virus and its interactions inside host cells, they suffer from the limitation that they cannot reproduce the symptoms of COVID-19 except in a specific cell type (in the case of cell lines) or organ (in the case of organoids).  Animal models solve this problem by allowing researchers to reproduce and observe the pathology of COVID-19 in both tissue-specific and systemic interactions.  A number of different animals have been identified to study the disease and to test potential therapeutic compounds.

A.   Small Animals

One group of researchers laid the groundwork for the discovery of animal models by conducting SARS-CoV-2 infection experiments using HeLa cells that expressed ACE2 proteins taken from multiple animal species, ranging from mice to humans.  Ironically, SARS-CoV-2 could use all ACE2 proteins except for mouse ACE2.  Researchers got around this by using transgenic mice that express human ACE2.  After infection with SARS-CoV-2, these mice showed weight loss, virus replication in the lungs, and interstitial pneumonia.  To find alternative small animal models, researchers performed molecular docking studies on the binding between ACE2 of various mammals and the S protein of SARS-CoV-2.  They discovered a suitable candidate in the Syrian hamster, which, when infected, displayed rapid breathing, weight loss, and alveolar damage with extensive apoptosis.

B.   Large Animals

Although small animals such as mice and Syrian hamsters have the advantage of reproducing at a faster rate, larger animal models are the preferred means of obtaining a faithful reproduction of COVID-19 pathology in humans.  One team of researchers reported nonlethal acute bronchiolitis in the lungs of a ferret model, while another team found that SARS-CoV-2 can replicate in ferrets and cats, but not pigs, chickens, and ducks.  The lesson learned is that ferrets and cats are preferable to rodents when selecting large experimental animals for the study of SARS-CoV-2.

Of the animals that are suitable for studying the pathology of COVID-19, the one currently the closest to humans in pathophysiology is the primate cynomolgus macaques.  When these animals were infected with SARS-CoV-2, the damage to type I pneumocytes led to pulmonary edema and the formation of hyaline membranes.  Researchers concluded from this that cynomolgus macaques can be infected with SARS-CoV-2 and reproduce some of the human pathologies of COVID-19.  

The use of a different primate, the rhesus macaque, in COVID-19 studies has been useful in confirming the therapeutic effects of adenovirus-vectored vaccine, DNA vaccine candidates expressing S protein, and remdesivir treatment.  

While the cynomolgus and rhesus monkey models are probably the best for replicating virus-human host interactions, they suffer from a major limitation: The reproduction rate in these primates is both lower and slower.  Hence this can be preceded by experiments with transgenic mice and Syrian hamsters.

III.         Conclusion

At the time this article was published on May 30, 2020, COVID-19 had spread rapidly throughout the world over the previous five months, with the number of infections and deaths showing no signs of abating.  There were no therapeutic prevention or intervention methods available, and the only way to control the pandemic and reduce the loss of life was to change people’s behavior through mask-wearing, quarantining, social distancing, and similar precautions.  At the time, therapeutic strategies for prevention and/or intervention were a desperate need.  

The article summarized here highlighted the need to conduct preclinical research on in vitroand model organisms at the same time as the clinical trials that were already under way.  As the article pointed out, it was necessary to undertake this research both to understand the virus and to test therapeutic agents for safety and efficacy.  The article provided important guidance to researchers in selecting appropriate cell and animal models for their SARS-CoV-2 research and in understanding the pros and cons of each model with an eye to developing still better ones.

IV.         Post-Script

In the 11 months since publication of the Takayama article, there has been an unprecedented, worldwide mobilization of resources to develop therapeutic agents and vaccines to slow and ultimately eradicate the virus and to prevent its re-emergence.  Today, even with the development and increasing use of numerous vaccines made by companies such as Moderna, Pfizer, and Johnson & Johnson, infection rates remain significant in many countries, most notably India and Brazil. While the United States still has the highest number of total cases and deaths of any country, it has among the 10 highest vaccination rates, with approximately 30% of the population fully vaccinated as of the end of April 2021.  Many other countries, particularly in Africa and Latin America, still have negligible vaccination rates, a stark reminder of the enormous disparities between rich and poor countries in reaping the fruits of vaccine research.  An estimated 83% of shots administered worldwide have been in countries having the highest incomes.  Only 0.2 percent of doses have been administered in low-income countries. 

Efforts to halt the spread of the virus have been complicated by the emergence of viral variants such as B.1.1.7, first described in the United Kingdom in the fall of 2020, and B.1.357 (South Africa) and P.1 (Brazil), all of which are believed to have higher rates of transmission and to be more virulent.  A large proportion of COVID-19 cases in the United States are attributable to these viral variants; for example, as of late March 2021, B.1.1.7 accounted for more than 44% of variants circulating in the United States.  Other variants are homegrown, believed to have originated in places such as Southern California (B.1.427, B.1.429) and New York (B.1.526).

More research is needed to determine how much more transmissible or virulent these variants are than their predecessors.  Recent studies have suggested that B.1.427/B.1.429 is at least 40% more effective at infecting human cells than some other variants of the virus.  Tests have also shown that people infected with B.1.427/B.1.429 can produce a viral load twice as large as that of people infected with other variants of the virus.  The bad news doesn’t stop there: A review of records from 308 coronavirus cases in San Francisco found that a larger percentage of people infected with the new variant had died compared to people infected with other variants.  Researchers have found that B.1.427/B.1.429 is not only more infectious, but also better at evading the immune system and current COVID-19 vaccines.  According to studies, antibodies from people who recovered from coronavirus infections involving other variants were less effective at blocking the B.1.427/B.1.429 in lab experiments.  Researchers saw the same results using blood serum from people who had been vaccinated for COVID-19. 

            Clearly, much research remains to be done.  The cell lines, organoids, and animal models detailed in the Takayama article may continue to play an important role in the search for new therapeutic agents and additional vaccines to halt the spread of the virus.

 

References:

Takayama, K. (2020). In Vitro and Animal Models for SARS-CoV-2 research. Trends in Pharmacological Sciences. 41(8), 513-517. Retrieved from 

 

Dowling, W. (2020). Animal models for COVID-19. Nature. 509-515

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DDD FOWLING, WILL