COVID and Animal Trials: A Systematic Review

Abstract

Extensive work is being done to form targeted drugs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; however, it is imperative to have a safe and effective vaccine against the same to win the war against this pandemic. For creating an efficacious vaccine, a proper animal model needs to be selected which can have an acceptable similarity of response as well as effects when administered to humans. For the present research, extensive search was conducted in MEDLINE and bioRxiv and medRxiv servers which were published in the English language from January 1, 2020, to August 20, 2020. Search terms included animal models, SARS-CoV-2, COVID-19, immune response against coronavirus, nonhuman primates, mice, ferrets, and macaques. In our study, creating an adequate immune response mimicking the response as in humans, as the endpoint, was considered as inclusion criterion while assessment of any additional therapies like safety as well as minimal tolerable dose using animal models as well as formation of adequate sample size of these models against COVID-19 was not considered. In our search, 163 articles were shortlisted, of them only 20 articles were finally included in our study which addressed to our inclusion and exclusion criterion. Our research articles focused on nonhuman primates, mice, hamsters, ferrets, cats, and dogs, with the main goal to investigate the role of animal models in the pathogenesis of COVID-19. It was evident in our research that animal models only mimic limited signs and symptoms experienced in COVID infection as compared to infections in humans. However, they are still essential to understand the pathogenesis, transmissibility of viral particles, and vaccine testing.

INTRODUCTION

Coronavirus pandemic has affected more than 400 thousand people around the world and has been causing severe respiratory syndromes, which also has been a cause for the mortality of the people affected. This disease has been commonly referred to as coronavirus disease 2019 or COVID-19.[1] Older people above 60–65 years and those having comorbidities are seriously affected and thus end up with multi-organ failure leading to death of the patient.[2] Spike protein binding is the main aspect which hastens the entry of virus particle inside human cells, which is facilitated by binding with angiotensin-converting enzyme-2 (ACE 2) protein on the host cell.[3] This increases the propensity of the infection, with mortality rate up to 5.8% with an average of 3.4% and covering almost 210 countries worldwide.[4,5] Hence, it is a matter of great concern that progress in the field of drug and vaccine development is increased at a breakneck speed. Pharmaceutical companies are trying to manufacture a potent vaccine by either using a weakened virus/or viral particle, viral RNA, or utilizing the target mechanism of spike protein, which allows entry of virus inside host cells.[6] An effective and prompt immune response against this virus is the endpoint considered in vaccine studies. Hence, to generate a high-level immune response, certain specific antigens are considered as the ideal candidate for vaccine production, but it requires host as well. This is where animal models come into the picture so that testing can be done easily without harming human lives.[7] However, currently, the animal models used for production of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are still under testing phase for many vaccine candidates, and more so the virus should induce similar pattern of disease and pathogenesis, as in humans, for successful vaccine generation.[8] Therefore, it is important to summarize if any of the animal models which are being currently used are turning out to be effective host reservoir.

METHODS

Search strategy and selection criteria

A systematic review was hence conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis guidelines[9] where studies which had laid emphasis on the generation of animal models against coronavirus infection were considered as the endpoint [Table 1].

Table 1.

Flow diagram illustrating the process of study selection. A systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta- analysis

Included studies

Researches where animal models were used for vaccine generation against COVID-19 infection were considered. Articles which were between January 1, 2020, and August 20, 2020 were included in the review.

Excluded studies

During the course of searching articles for our systematic review, the articles where only abstracts were available and the ones which were based on SARS and Middle East respiratory syndrome (MERS) as primary infection were excluded from our search criteria. Articles which were published in languages other than English were also excluded.

Data extraction

Data from articles were extracted individually by two reviewers, which were used to construct the tables.

Quality assessment

Quality of each publication was evaluated by two independent reviewers. This review addressed various domains: vaccine production, quality of immune response, active immunity, dose of administration, and pathogenesis of the disease in animal models.

Assessment of risk of bias and applicability in included studies

Assessment of risk of bias and its inclusion in the studies were comprehended by reviewers independently. SYRCLE's risk of bias tool was used for quality evaluation of animal studies and Nature Publication Quality Improvement Project score sheet was used to assess in vitro studies.[10,11]

RESULTS

During our search on MEDLINE and various other Internet platforms and servers, we identified 69 studies and 94 preprints, of which 143 articles were excluded as many were not original articles, not related to COVID-19 infection. Only 20 articles were consistent with our inclusion and exclusion criterion. Our research articles focused on nonhuman primates, mice, hamsters, ferrets, cats, and dogs, with the main goal to investigate the role of animal models in pathogenesis of COVID-19 infection, risk of transmission, rate of infectivity, and inoculation with SARS-CoV-2 viral strains with ascending doses and studying the therapeutic effects [Table 2].

Table 2.

Summary of studies using nonhuman primate models of COVID-19

Species and concerned studies	Number of samples (n)	Outcome measures
Rhesus macaques
Munster et al. (2020)	8	Pathogenesis of COVID-19
Yu et al. (2020)	2	Pathogenesis of COVID-19 in aging animals
Van Doremalen et al. (2020)	6	Evaluation of DNA vaccine
Gao et al. (2020)	4	Evaluation of an inactivated vaccine
Williamson, B.N. et al. (2020)	6	Testing of antiviral therapy
Chandrashekar et al. (2020)	9	Immune protection after a second exposure
Bao et al. (2020)	7	Immune protection after a second exposure
Deng, W. et al. (2020)	5	Viral infection routes
Mice
Boudewijns et al. (2020)	20	Interferon response to SARS-CoV-2 Infection
Bao et al. (2020)	15	Pathogenesis of COVID-19
Lv et al. (2020)	10	Cross-reactivity of antibodies against SARS-CoV and SARS-CoV-2
Pruijssers et al. (2020)	10	Establishment of mouse-adapted SARS CoV-2 model of COVID19
Dinnon et al. (2020)	33	Evaluation of vaccine and therapy in mouse-adapted SARS-CoV-2 model
Ferret
Kim et al. (2020)	6	Viral infection and transmission
Golden Syrian Hamster
Chan et al. (2020)	10	Study of pathogenesis, therapeutics, and vaccines
Sia et al. (2020)	6	Immunological studies for vaccine development
Cynomolgus macaques
Lu et al. (2020)	6	Comparisons of pathogenesis between COVID-19, SARS-CoV, and MERS CoV
Rockx et al. (2020)	10	Comparisons of pathogenesis between COVID-19, SARS-CoV, and MERS CoV
Finch et al. (2020)	6	Evaluation of medical interventions
African green Monkey
Woolsey et al. (2020)	6	Pathogenesis of COVID-19

SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2, MERS CoV: Middle East respiratory syndrome coronavirus

Rhesus macaques

In case of rhesus species, they have a similar amount of ACE-2-binding receptor proteins on their cells. Hence, they turn out to be near ideal choice for testing of vaccines.[12] In their case, pulmonary infection is present when inoculated with SARS-CoV-2 virus and can be verified with the help of radiographs. They also have an increased number of viral particles that can be extracted from their nose and throat samples.[13]

Ferrets

Ferret models are similar to human lungs as they are susceptible to coughing and sneezing reflexes, so they can be used to test COVID-19 infection also.[14] They also have high transmissibility rates, as ferrets can spread the virus with the help of direct contact and/or aerosols.[15]

Mice

Laboratory mice is always an effective and much more cost-effective and easy to handle animals for vaccine testing. Recently, humanized mice versions which have similar ACE2 numbers have also been created to successfully test SARS-CoV-2 infection.[16]

Syrian hamster

This animal model can also be used for testing as they have similar phenotypic alterations in ACE-2-binding mechanisms as and when compared to human binding sites on the cell membrane, which is an essential requisite for the pathogenesis of COVID infection.

DISCUSSION

Many animal models have recently been included in various studies which have proved to be hopeful for effective vaccine generation. Moderna, a USA-based company, has already completed its phase I trials successfully which helps in safety as well as toxicity of these vaccine targets in humans.[6] Pfizer is already in phase III trials and according to some, the vaccine generated by them is around 90% effective. Many studies of animal models have been reviewed in connection with SARS and MERS infection, these models usually failed to replicate the conditions which are similar to the human environment for trail and testing of vaccines. In addition, the results of this systematic review are also consistent with the above findings when COVID-19 infection is considered.[17] However, viral titers which were high in number were discovered in the respiratory passage, and many mild symptoms were similar to the symptoms that humans experience. Unfortunately, the common complications like acute respiratory distress syndrome (ARDS) and coagulopathy, which is common in human COVID patients, were not replicated in any of these animal models.[18] Most cases that end up in intensive care units have hypoxemia which is caused due to ARDS and coagulopathy leads to severe thrombo-embolic complications in humans, even in the young age population.[19,20] The postmortem studies of the individuals who died of these complications revealed extensive hyalinization and inflammatory cell damage leading to destruction of the alveolar air spaces which are essential,[21] further supported by the fact that in these individuals, many micro- and macrothrombi were noted in the lung tissue which compromised their lung perfusion leading to higher mortality.[22] A full-blown COVID infection in humans significantly differs from these animal models, as the severity of respiratory as well as thrombo-embolic manifestations is not simulated to near ideal situations. However, mechanism of respiratory symptoms as well as pathogenesis is not fully clear as many pathways may lead to an increase of tissue factor which will cause endothelial injury and hence thrombotic episodes with complement activation, which further compromises the vascular system, especially in the pulmonary area which further worsens the symptoms by activating the clotting cascade and subsequent formation of thromboemboli. Hence, animal models which can simulate these conditions will help in understanding the pathogenesis better.[23,24,25,26,27] Rhesus macaques and mice were used to test both the antiviral medications and vaccine candidates where the medication stopped viral replication leading to a recovery in case of pneumonitis.28 In case of vaccine candidates, there was an increase in titer of anti-COVID antibodies as well as a decrease in the viral load which helped in preventing respiratory infection significantly.[29] This leads to a promising outcome for vaccine efficacy as well as antiviral medication effectiveness against SARS-CoV-2. Unfortunately, even after the epidemics such as SARS and MERS, scientists have not been able to form an effective animal model to create conditions appropriate for the spread of COVID-19 in animals.[30,31] Primates have similar binding affinity to COVID-19 virus as when related to humans,[32] which differs in stark contrast to other animals such as hamsters and mice, which have low-to-medium affinity. This issue has been seen in many studies where mice does not support increased SARS-CoV-2 replication as compared to a chimera as the former does not have more amount of ACE-2-binding protein receptors on their cells.[33] Recently, complex phenotype of COVID-19 was found with the help of single-cell RNA genomic sequence technology which might help us to decide on the dissimilarities between primate and nonprimate species-specific infections.[34] For proper entry of viral particles inside the host cell, it is imperative to study the variations encountered in the distribution of ACE-2-binding protein and TMPRSS2, as the amount of these receptors as well as their surface configuration varies in different organs as well as between two species also. In the primate lung when compared with human pneumocytes, ACE-2 expression was lower in the animals, especially in type II pneumocytes.[35] During the course of evolution, lot of modifications have happened, which has led to the limitation of this viral infection in only the respiratory system of these animals.[36] This explains that animal models show less of symptoms as compared to humans, which poses a practical challenge in vaccine development for humans in case of COVID-19 infection. Our systematic review did have its share of limitations, as we had included preprints in the studies which were not peer-reviewed as yet. We also did have less number of articles studied, and thus, the scope of this study needs to be widened.

CONCLUSION

Through this systemic review, we were able to find out that animal models only mimic limited signs and symptoms experienced in COVID infection as compared to infections in humans. However, they are still essential to understand the pathogenesis, transmissibility of viral particles, and vaccine testing. Hence, an animal model should be selected carefully, which can help outlining the vaccine testing strategy effectively.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.