Skip to main content
Advertisement
  • Loading metrics

Advances and gaps in SARS-CoV-2 infection models

Abstract

The global response to Coronavirus Disease 2019 (COVID-19) is now facing new challenges such as vaccine inequity and the emergence of SARS-CoV-2 variants of concern (VOCs). Preclinical models of disease, in particular animal models, are essential to investigate VOC pathogenesis, vaccine correlates of protection and postexposure therapies. Here, we provide an update from the World Health Organization (WHO) COVID-19 modeling expert group (WHO-COM) assembled by WHO, regarding advances in preclinical models. In particular, we discuss how animal model research is playing a key role to evaluate VOC virulence, transmission and immune escape, and how animal models are being refined to recapitulate COVID-19 demographic variables such as comorbidities and age.

In February of 2020, the World Health Organization (WHO) R&D Blueprint convened a group of experts to develop preclinical models of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Since its inception, the goal of this WHO COVID Modeling group (WHO-COM) has been to accelerate the development of Coronavirus Disease 2019 (COVID-19) vaccines and therapeutics by rapidly sharing data among member scientists worldwide. In addition, concerns were raised at that time about the possibility of vaccine-associated enhanced respiratory disease (VAERD) or antibody-dependent enhancement (ADE) after vaccination or infection. In September of 2020, the WHO-COM published a review on COVID-19 animal models [1], which reflected the state-of-the art at that time, with the vast majority of publications authored by members of the group.

Preclinical studies in nonhuman primates (NHPs) of COVID-19 vaccines that are currently being deployed [25] proved remarkably predictive of the outcome of clinical efficacy studies. In particular, NHP studies not only predicted high clinical efficacy of these vaccines but also suggested immune correlates of protection. Moreover, preclinical studies accurately predicted that protection against severe pneumonia would be easier to achieve than protection against viral replication in nasal mucosa. These observations confirm the value and importance of the use of animal models for COVID-19.

In 2021, with several vaccines rolling out worldwide and the detection of variants of concern (VOCs), the development of preclinical models of SARS-CoV-2 infection and their role in COVID-19 research has entered into a new phase. This paper provides an update from the WHO-COM regarding advances in preclinical models. In particular, we discuss how animal model research has provided insight into VOC pathogenesis and correlates of protection and has helped therapeutic development. Finally, we discuss the current status of VAERD research and the race to develop models that recapitulate COVID-19 demographic variables such as comorbidities and age.

Animal models to study VOCs

As the current pandemic evolves, several virus variants carrying multiple mutations have emerged in different regions of the world. Some variants have been classified by WHO as variants of interest (VOIs) or VOCs, based on epidemiological evidence of enhanced transmission and possible evasion from natural and vaccine-induced immunity [6]. Animal models have a key role in the evaluation of VOC transmission, immune escape, and pathogenicity.

Vaccine cross-protection and transmission

Recent studies in mice, hamsters, and NHPs show that animals previously infected or vaccinated against lineage A SARS-CoV-2 (for example, the original Wuhan strain) [7] are protected against challenge with homologous as well as heterologous virus strains including the alpha (B.1.1.7), beta (B.1.351), gamma (B.1.1.28.1), and delta (B.1.617.2) VOCs [814]. In the NHP model, however, more viral breakthroughs were observed following beta VOC challenge as compared with homologous WA1/2020 challenge [12,15]. In addition to protection against disease, another concern was to determine whether reinfection with VOCs resulted in SARS-CoV-2 shedding, which would raise the possibility that asymptomatic reinfected individuals might transmit VOCs. In this regard, hamsters reinfected with VOCs were indeed shown to shed SARS-CoV-2 for a number of days [8,14,16]. However, transmission studies performed in cats indicated that infected animals did not shed enough virus for transmission to cohoused naïve sentinel cats [17]. These results are in agreement with the finding that, although vaccinated individuals can be reinfected, transmission of delta VOC from these individuals may be substantially reduced in comparison with nonvaccinated subjects [18]. Importantly, virus shedding in hamsters, ferrets, and NHPs was reduced by intranasal vaccination [19,20], showing perhaps an added value of mucosal vaccines to control VOC expansion.

It is now clear, however, that, in competition studies, at least the alpha and beta VOCs show enhanced transmission in comparison with lineage A SARS-CoV-2 in a number of models, including hamsters, ferrets, and white-tailed deer [2124]. This attribute could be dependent, at least partially, on the presence of VOC-specific spike substitutions such as N501Y, D614G, and V367F, which improve the affinity of the SARS-CoV-2 spike protein for the human and hamster angiotensin-converting enzyme 2 (ACE2) receptors. The aromatic N501Y substitution that is present in the alpha, beta, and gamma VOC is associated with increased transmission in humans but also allows for infection in the wild-type mouse using the mouse ACE2 receptor [25]. Thus, 2 recent studies have shown that wild-type mice are susceptible to certain SARS-CoV-2 VOCs, specifically to the beta (B.1.351) and gamma (P1) VOCs [26,27], most likely due to the N501Y substitution. Although mild lesions and viral replication were observed in nasal turbinates and lung of these wild-type mice inoculated with these VOCs, no significant clinical signs were observed. These results open the avenue to use wild-type mice as a potential animal model of asymptomatic infection with SARS-CoV-2, mainly to study immune responses, for which laboratory reagents are widely available. More threatening, these observations also raise concerns on the possibility of interspecies transmission, with new variants expanding their tropism toward other animal species resistant to the ancestral viral strains and, eventually, becoming novel secondary viral reservoirs [28]. However, the highly relevant delta variant has a different mutation pattern that does not include an N501Y substitution, but a P to R substitution in the spike protein cleavage site and therefore may react differently in the animal models than other VOCs. This raises the possibility that SARS-CoV-2 could also evolve into a human-specific virus with reduced cross-infective properties in other mammals. In this regard, human tissue culture microfluidic systems have been developed to supplement the infection modeling landscape [29,30] and may offer a scalable solution to such a scenario if it were to emerge. Characterization in the full spectrum of available models and systems and with different delta VOC isolates is therefore suggested. However, it must always be kept in mind that the emergence of additional new VOCs will lead to a time delay in testing in animal models, since the selected viruses must first be isolated and characterized in vitro and subsequently distributed to the different laboratories worldwide.

VOC pathogenesis in animal models

Importantly, even though VOCs readily infected the lower respiratory tract of hamsters and NHPs, none of these variants seemed to show enhanced virulence in these animal models, although increased production of proinflammatory cytokines was observed in hamsters infected with the alpha VOC [9]. In a comparative study carried out in rhesus macaques, infection with the beta VOC resulted in lower clinical scores and lower levels of virus replication in comparison with ancestral B.1 virus and alpha VOC [31]. These findings were confirmed in a direct comparison between B.1 and alpha VOC infection in African green monkeys [32]. Conversely, studies in other models such as transgenic mice expressing the human ACE2 (huACE2) receptor driven by the cytokeratin-18 (K18) gene promoter (K18-huACE2) have yielded different outcomes. In these mice, infection with the beta VOC resulted in enhanced infectivity and a quicker disease progression in comparison to one of ancestral variants (B.1) [26]. Such enhanced infectivity could be due, at least to some extent, to the expression of higher levels of interferon antagonist proteins by some VOCs [33]. Other studies have, however, shown reduced fitness of the beta VOC in mice in competition trials [22] (Fig 1). One possible explanation for differences in VOC virulence when comparing results in different animal models is the rise of mutations leading to increased processing and fusion by the S protein of SARS-CoV-2 [34,35]. As VOC phenotypic features appear dependent on interactions with host factors, experimental demonstration of enhanced virulence or transmission with emerging VOC may depend on the animal model and the specific VOC strain used. One strategy to minimize the variables during experiments has been the use of syngeneic viral backbones generated by reverse genetics [36]. This approach demonstrated the fitness advantage of S-614G versus S-614D in a syngeneic direct competition assay [21] and recently also demonstrated the role of the spike protein of the alpha VOC for enhanced transmission properties in a transgenic mouse model [22]. It is important to consider that various experimental setups and the use of different VOCs could affect the experimental outcome. A series of experiments in different animal models and with different VOCs are necessary for particularly robust conclusions.

thumbnail
Fig 1. SARS-CoV-2 VOC in animal models.

The schematic summarizes findings related with virulence, transmission, and cross-protection gathered in the indicated animal models so far. Figure created with Biorender (Biorender.com). huACE2, human angiotensin I-converting enzyme 2; K18, cytokeratin-18; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; VOC, variant of concern.

https://doi.org/10.1371/journal.ppat.1010161.g001

Vaccine-associated enhanced respiratory disease

Vaccine-associated enhanced disease (VAED) defines adverse events that affect individuals infected with a pathogen after receiving a prior vaccine against that same pathogen. More specifically, VAERD was observed in individuals immunized with formalin-inactivated vaccines against measles or respiratory syncytial virus (RSV) decades ago [37,38]. ADE, on the other hand, is a phenomenon where preexisting vaccine or infection-induced antibodies enhance infection of FcγR expressing target cells, which results in increased disease. ADE is well documented to occur after secondary infections with dengue viruses belonging to different serotypes [39].

There were initial concerns that SARS-CoV-2 vaccines, in particular whole-inactivated vaccines, might lead to VAERD or ADE. These concerns were heightened by in vitro evidence of ADE for SARS-CoV and Middle East Respiratory Syndrome (MERS)-CoV [40], experimental data indicating eosinophilic VAERD in studies of SARS-CoV and MERS-CoV vaccines in mice [41,42], and hepatitis observed in a SARS-CoV ferret model [43]. These concerns led to the assessment for possible VAERD and ADE by the WHO-COM group in different animal models of SARS-CoV-2 pathogenesis.

These initial concerns have been now alleviated by several findings in animal models. First, all vaccines that either are emergency use authorized or in late clinical development phases induce strong T-helper 1 (Th1) CD4-mediated responses, which are associated with high ratios of neutralizing versus nonneutralizing antibodies and reduced risk of VAERD [44]. Moreover, several experiments performed by WHO-COM scientists have utilized experimental alum-adjuvanted and formaldehyde-inactivated whole virus vaccines and subsequent SARS-CoV-2 challenge to address any evidence of VAERD. In rhesus macaques, histopathological analysis revealed no evidence of enhanced lung pathology, and a rapid rise in neutralizing antibodies was seen after challenge [45]. In hamsters and ferrets, on the other hand, a mild increase of lung pathology was observed at 5 days and 7 days postinoculation (dpi), respectively, in comparison to unvaccinated controls. Nonetheless, in both models, pathology was reduced at 13 dpi to comparable levels for vaccinated and unvaccinated animals. The enhanced pathology was characterized by increased perivascular cuffing (ferrets and hamsters) and greater influx of mononuclear cells and granulocytes in alveoli with thickening of alveolar wall, proliferation of type II pneumocytes, and hemorrhages (hamsters). No clear influx of eosinophils was observed in either species. Noteworthy, hamsters showed no neutralizing antibodies post-immunization and no protection against challenge, but lung cytokines were markedly skewed toward Th2 [45]. In addition, a recent study in K18-hACE2 mice immunized with a very impure formalin-inactivated SARS-CoV-2 preparation and an aluminum hydroxide-based adjuvant demonstrated earlier onset of SARS-CoV-2 replication and disease in comparison to the naïve control groups or mRNA-vaccinated animals [46].

As VAERD typically develops after vaccine-induced antibody responses wane, it may be too soon to conclude about the presence of VAERD in SARS-CoV-2 vaccination. However, the data currently available from animal experiments, showing the absence of VAERD in NHPs and a transient increase of lung pathology in ferrets and hamsters, are reassuring. Moreover, VAERD has not been reported in humans immunized with inactivated SARS-CoV-2 vaccine preparations.

Animal models and VOC prophylaxis and therapy

Despite the rapid and successful development of COVID-19 vaccines, unequal global vaccine distribution has contributed to the rise of VOCs with potential to escape natural as well as vaccine-induced immunity. This, together with the lack of medical countermeasures against severe COVID-19, has resulted in increased efforts to develop prophylactic and therapeutic strategies against SARS-CoV-2 infection, for which animal models are playing a key role. Although revising all therapeutic strategies against COVID-19 is out of the scope of this study, we would like to point out recent preclinical studies with potential to treat infections caused by VOCs. Of these, the use of polyclonal antibodies or antibody cocktails against the SARS-CoV-2 spike receptor binding domain (RBD) provide an advantage by binding to multiple epitopes, thereby reducing the chance of VOC immune escape. Such strategies have shown potent prophylactic and therapeutic activity in mouse models of infection as well as hamsters [47,48]. Alternatively, monoclonal antibodies such as COVA1-18 and P5C3 with broad neutralizing activities have also shown prophylactic effect in hamsters, hACE2 mice, and cynomolgus macaques and potent reduction of virus replication postexposure [49,50]. P5C3 has also shown neutralization of all known VOCs to date at picomolar concentrations [50]. Neutralizing single domain antibodies (nanobodies) can be administered intranasally and have also shown to greatly reduce SARS-CoV-2 replication in hamsters [51,52]. A complementary strategy against VOC could be the development of antibody cocktails with enhanced Fc-mediated functions, which have been shown to greatly contribute to SARS-CoV-2 humoral immunity [53]. Consequently, anti-SARS-CoV-2 monoclonal antibodies with optimized Fc domains have shown potent prophylactic and therapeutic activity in several mouse models and hamsters [54].

In the early days of the pandemic, animal models were also key for testing nucleoside analog drugs such as remdesivir [55] and favipiravir [56]. More recently, molnupiravir (MK-4482) has shown great potential as an orally available drug, which significantly reduced SARS-CoV-2 replication in hamsters [57] and prevented transmission in ferrets [58]. Nucleoside analogs such as favipiravir have also shown synergistic activity in combination with other drugs such as 3CL protease inhibitors in huACE2 mice [59]. Thus, animal models may be needed to test combination drugs against COVID-19. For this, as we will discuss below, the development of more severe models of disease may be needed.

The search for an animal model of severe SARS-CoV-2 disease

One of the main gaps still remaining in the development of SARS-CoV-2 infection models is the identification of a preclinical animal model that recapitulates the severe and lethal form of human COVID-19. Such a model would be of great advantage for several aspects of research. First, it would provide a tool to study the transition from mild to severe disease, which would possibly lead to the identification of disease mechanisms and biomarkers. Second, it would expand the presently available animal models to evaluate vaccines and therapeutics, which could result in urgently needed rescue medical countermeasures. Since the first review published by the WHO-COM [1], the hamster model has emerged as the one that more closely recapitulates moderate disease in humans. Hamsters not only develop respiratory disease after SARS-CoV-2 infection, but also display some other important clinical hallmarks in patients such as anosmia, neurotropism, and vascular inflammation [6062]. With some exceptions, most laboratories have observed that hamsters tend to lose weight rapidly after experimental infection with SARS-CoV-2 reaching 5% to 20% or more of body weight loss at the peak of infection. However, in some parts of the world, ethical approvals establish euthanasia endpoints at 20% to 25% of weight loss, while in other laboratories, animals are sometimes allowed to lose 30% of body weight. These regulatory differences complicate the definition of severe disease in animal models of infection. Nevertheless, pathological examination of infected hamster lungs shows evidence of severe interstitial pneumonia with high levels of bronchoalveolar damage and inflammation. Unfortunately, the hamster model presents a few unresolved challenges; for example, there is a need to better understand the relationship between the severity of pulmonary pathology and the mild to moderate clinical signs. In order to address this, it seems imperative to develop more tools to study hamster immunology, for example, antibody panels for multiparametric flow cytometry. Moreover, male hamsters show more severe lung lesions than females and less efficient antibody responses [63,64]. In the absence of comorbidities or coinfections, an explanation for these findings is likely dependent on sex-associated differences in immune responses. Indeed, male COVID-19 patients have shown higher levels of proinflammatory cytokines and reduced T cell–mediated immunity in comparison with female patients [65]. Whether this is the case also in the hamster model again depends on the development of more advanced tools to study hamster immunology.

Furthermore, the disease development may also be related to age of the used hamsters with older hamsters showing more severe disease progression [66], although others found no substantial age-related difference [67,68]. Despite the gaps of knowledge, hamsters have become the model of choice for preclinical testing of vaccines and therapeutics together with different species of NHPs [6972].

In addition to the hamster model, several murine models of severe disease are now available, including infection of common laboratory strains with mouse-adapted SARS-CoV-2 and certain SARS-CoV-2 VOCs and infection of mice expressing huACE2 transgenically or of mice with “knock-in” of huACE2 [7376]. Mice develop pathological signs of pneumonia that range from mild to severe. In some instances, mice also develop anosmia, a common manifestation of the human COVID-19 [76]. Mice have the advantage of well-characterized genetics and the availability of mice that are completely or conditionally deleted in genes of interest. In addition, the vast existence of reagents to study immune responses in mice also allows a much better characterization of the immunology related to SARS-CoV-2 compared to hamsters or ferrets.

Finally, as discussed above, several monoclonal antibodies and small molecule antivirals against SARS-CoV2 [9] are in development, some of which are approved in the United States or may receive market authorization elsewhere in 2021. An important open question is whether escape or resistant virus variants may emerge against either of these therapies. The current SARS-CoV2 animal infection models typically show a limited duration of virus replication and are therefore not well suited to explore whether, in particular with suboptimal doses of either a monoclonal antibody or a small molecule antiviral, drug-resistant variants may emerge. It will therefore be important to develop SARS-CoV2 infection models in which the virus replicates to sufficiently high titers for extended periods of time without causing severe pathology that would require early euthanasia of these animals. To this end, either strains of animals with immunodeficiencies or the experimental induction of immunodeficiencies by pharmacological interventions would be worth exploring.

Age and comorbidities

In human COVID-19 disease, there is a strong association of severe disease with age and/or preexisting comorbidities including cardiac disease, diabetes/obesity, hypertension, and chronic respiratory diseases. For COVID-19, age is the best correlate of poor outcome, with those over the age of 85 having a 630-fold increase in death compared to those 18 to 29 years old (https://www.cdc.gov/coronavirus/2019-ncov/covid-data/investigations-discovery/hospitalization-death-by-age.html). Refinement of preclinical models to recapitulate the effect of age and comorbidities in SARS-CoV-2 infection has been an important effort during the last year.

Age

Mouse models were one of the first options to explore the effect of age in SARS-CoV-2 infection. There are numerous mouse models of aging that could be applied to COVID-19 studies. The main strategy has been to cross these mouse models with huACE2 transgenic mice or to use mouse-adapted virus. Overall, these studies indicated that the severity of SARS-CoV-2 infection in C57BL/6 and Balb/c mice is age dependent [73]. Indeed, young mice were resistant to SARS-CoV infection even if they lacked IFN-I expression or the capacity to mount adaptive immune responses [77], while aged mice infected with SARS-CoV-2 showed greater weight loss, clinical signs, and pathology than their young counterparts despite comparable viral loads [73]. Mechanistically, the positive correlation between age and severity in SARS-CoV–infected mice was associated with increased age-dependent inflammation in the lung [78], which is consistent with findings in humans [79].

The effect of age in SARS-CoV-2 infection has been also evaluated in ferrets. These studies showed that 1- to 2-year-old ferrets had prolonged fever compared to young ferrets, which developed little-to-no fever. Three-year-old ferrets had fevers that lasted out past 10 dpi. In addition, older ferrets lost more weight upon infection and regained it more slowly than younger ferrets [80]. More severe lung pathology also was noted in the older ferrets at 5 dpi. Of note, older ferrets also had higher viral titers in nasal washes and fecal specimens for a longer period and were more likely to transmit the virus to younger ferrets. This is also consistent with findings in older patients, in which immune senescence, loss of type I IFN responses, decline in antigen presentation, and reduced T cell responses have shown to delay viral clearance [81,82].

This correlation between SARS-CoV-2 infection severity and age has been also confirmed in the NHP model. A study comparing rhesus macaques and baboons suggested that viral pneumonia may persist longer in older animals of either species, and a reduction in antibody responses in aged rhesus macaques [83]. Aged rhesus macaques also have been shown to have increased rectal shedding, slower viral clearance, higher viral loads, more severe lung pathology, higher levels of proinflammatory cytokines, and greater body weight loss [84]. Increased shedding of viral RNA from the upper respiratory tract was observed in older cynomolgus macaques in one study, but this was not associated with increased disease severity [84]. A multiomics study comparing subadult and aged rhesus macaques showed that age did not substantially affect acute disease; however, an age-specific divergence of immune responses emerged during the postacute phase of infection (7 to 21 dpi). As in humans, advanced age resulted in a delayed or impaired induction of antiviral cellular immune responses and a delay in the efficient return to immune homeostasis [85].

Comorbidities

Less is known about the effect of comorbidities during SARS-CoV-2 infection in animal models. The reason is that, with the exception of mice, comorbidities are difficult to model in animal experiments. In mice, however, the huACE2 model can be crossed with other specific models of disease such as diabetes, chronic inflammation, or cardiovascular disease (CDs) models. Alternatively, mouse-adapted SARS-CoV-2 or any of the VOCs containing the N501Y spike protein mutation can be directly used to infect mice. Finally, huACE2 can be also expressed to mouse models of disease via adenovirus delivery. The latter approach has been used to study the effects of CDs and diabetes mellitus in SARS-CoV-2 infection and has shown that preexisting CDs resulted in enhanced inflammation and risk of myocardial injury upon infection [86], which is consistent with observations in humans [87]. Another strategy is the induction of comorbidities, in particular diabetes and obesity, through changes in rodent diet. Thus, diet-induced obesity in mice resulted in more severe disease upon infection with N501Y-containing SARS-CoV-2 [25]. Similarly, hamsters fed a western diet for 16 weeks showed greater weight loss and higher viral loads, as well as prolonged viral shedding compared to controls fed a regular rodent diet [88]. Despite these differences, there were no significant effects of obesity in respiratory function or SARS-CoV-2–induced pathology between the 2 groups. Other comorbidities associated with severe disease in COVID-19 have not yet been studied in rodents.

Correlates of protection

Establishing the immunological correlates of protection remains a key question for vaccine deployment and evaluation. While high levels of neutralizing antibodies in sera likely are associated with protection against disease, the contribution of cellular immunity to protection, including T cells and Fc-dependent effector antibody functions, remains uncertain. In fact, in Phase III SARS-CoV-2 mRNA and Ad26 vaccine clinical trials, protection against clinical infection could be seen even before the appearance of protective antibody titers in sera [89]. Moreover, correlates of protection against clinical disease and against asymptomatic infection and transmission likely differ. The establishment of robust correlates of protection is necessary to allow immunobridging, which extrapolates vaccine immunogenicity in humans to a protective effect, based on the immunogenicity and protection observed in animal models. Immunobridging will benefit from standardized immunological assays to quantify correlates of protection across animal experiments and clinical studies (Fig 2). It has been used for the expansion of mRNA vaccine approval to younger age groups [90] and will likely expedite the approval of second-generation vaccines without costly and lengthy efficacy studies. It will also help in determining whether and when boosting is needed, and whether vaccine strain updates are needed. One caveat might be that different vaccines might have different correlates of protection, making a universal correlate of protection difficult to achieve. For example, from NHP studies, it has been suggested that T cells can contribute to protection when antibody levels are suboptimal, or start to wane [91]. This indicates that correlates for long-term protection for an individual may change over time or may include multiple immune parameters. Therefore, it will be important to define immune thresholds for protection. These thresholds can be different for different target groups (the very young, the elderly, pregnant, immune-compromised, or people with comorbidities), which is currently being investigated in the available animal models that reflect the physiological and immunological status of these target groups. Meta-analyses of data generated in clinical trials and postapproval are crucial to validate the correlates of protection identified in preclinical studies.

Animal models also might be valuable to validate correlates of protection from infection, disease, and transmission. Passive antibody studies in multiple animal models have demonstrated that high titers of polyclonal or monoclonal neutralizing antibodies can protect from disease. Similarly, T cell depletion experiments and adoptive transfer experiments analogously can address the contribution of T cell immunity in protection from disease in mice and NHPs.

thumbnail
Fig 2. Animal models and immunobridging.

Comparative and standardized studies in animal models such as those performed by WHO-COM scientists can help to extrapolate vaccine immunogenicity data across preclinical and clinical studies. Figure created with Biorender (Biorender.com). VAERD, vaccine-associated enhanced respiratory disease; VOC, variant of concern; WHO-COM, WHO COVID-19 Modeling group.

https://doi.org/10.1371/journal.ppat.1010161.g002

Conclusions and future course

Animal models for SARS-CoV-2 infection have fostered the development of COVID-19 vaccines and therapeutics during the first year of the pandemic, several of which have been deployed on a global scale. Going forward, animal models can still fill important knowledge gaps. For example, preclinical animal studies will be important to understand disease progression and identify biomarkers that can aid us better predict the course of human disease. Animal model studies likely will allow the experimental validation of predicted correlates of protective or dysregulated immunity in humans. Similarly, animal models will be essential to evaluate VOC pathogenicity and transmissibility and to further assess the potential risk of VAERD, especially in the context of heterologous vaccination regimens. It will also be essential to develop SARS-CoV-2 animal infection models in which the virus replicates for extended periods of time, thus allowing for assessment of emergence of resistant variants against vaccines or therapies. Finally, as there is still much to learn about the role of comorbidities in human COVID-19, animal models with comorbidities will be needed to dissect the role of infection versus comorbidity in disease severity. These efforts may also lead to the design and evaluation of specific therapies against severe COVID-19 that function best in the background of particular medical conditions.

References

  1. 1. Munoz-Fontela C, Dowling WE, Funnell SGP, Gsell P-S, Riveros-Balta AX, Albrecht RA, et al. Animal models for COVID-19. Nature. Nature Publishing Group; 2020;586:509–15. pmid:32967005
  2. 2. Mercado NB, Zahn R, Wegmann F, Loos C, Chandrashekar A, Yu J, et al. Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques. Nature. Nature Publishing Group; 2020:1–11. pmid:32731257
  3. 3. van Doremalen N, Lambe T, Spencer A, Belij-Rammerstorfer S, Purushotham JN, Port JR, et al. ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques. bioRxiv. Cold Spring Harbor Laboratory Preprints. 2020. pmid:32511340
  4. 4. Corbett KS, Flynn B, Foulds KE, Francica JR, Boyoglu-Barnum S, Werner AP, et al. Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates. N Engl J Med. 2020:NEJMoa2024671. pmid:32722908
  5. 5. Vogel AB, Kanevsky I, Che Y, Swanson KA, Muik A, Vormehr M, et al. BNT162b vaccines protect rhesus macaques from SARS-CoV-2. Nature. Nature Publishing Group; 2021;592:283–89. pmid:33524990
  6. 6. Krause PR, Fleming TR, Longini IM, Peto R, Briand S, Heymann DL, et al. SARS-CoV-2 Variants and Vaccines. N Engl J Med. Massachusetts Medical Society; 2021;385:179–86. pmid:34161052
  7. 7. Rambaut A, Holmes EC, O’Toole Á, Hill V, McCrone JT, Ruis C, et al. A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nat Microbiol. Nature Publishing Group; 2020;5:1403–7. pmid:32669681
  8. 8. Yinda CK, Port JR, Bushmaker T, Fischer RJ, Schulz JE, Holbrook MG, et al. Prior aerosol infection with lineage A SARS-CoV-2 variant protects hamsters from disease, but not reinfection with B.1.351 SARS-CoV-2 variant. Emerg Microbes Infect. Taylor & Francis; 2021;10:1284–92. pmid:34120579
  9. 9. Abdelnabi R, Boudewijns R, Foo CS, Seldeslachts L, Sanchez-Felipe L, Zhang X, et al. Comparing infectivity and virulence of emerging SARS-CoV-2 variants in Syrian hamsters. EBioMedicine. 2021;68:103403. pmid:34049240
  10. 10. Nuñez IA, Lien CZ, Selvaraj P, Stauft CB, Liu S, Starost MF, et al. SARS-CoV-2 B.1.1.7 Infection of Syrian Hamster Does Not Cause More Severe Disease, and Naturally Acquired Immunity Confers Protection. mSphere. 2021:e0050721. pmid:34133199
  11. 11. Hassan AO, Shrihari S, Gorman MJ, Ying B, Yaun D, Raju S, et al. An intranasal vaccine durably protects against SARS-CoV-2 variants in mice. Cell Rep. 2021;36:109452. pmid:34289385
  12. 12. Yu J, Tostanoski LH, Mercado NB, McMahan K, Liu J, Jacob-Dolan C, et al. Protective efficacy of Ad26.COV2.S against SARS-CoV-2 B.1.351 in macaques. Nature. Nature Publishing Group; 2021;596:423–27. pmid:34161961
  13. 13. Tostanoski LH, Yu J, Mercado NB, McMahan K, Jacob-Dolan C, Martinot AJ, et al. Immunity elicited by natural infection or Ad26.COV2.S vaccination protects hamsters against SARS-CoV-2 variants of concern. Sci Transl Med. American Association for the Advancement of Science; 2021. pmid:34705477
  14. 14. Mohandas S, Yadav PD, Shete A, Nyayanit D, Sapkal G, Lole K, et al. SARS-CoV-2 Delta Variant Pathogenesis and Host Response in Syrian Hamsters. Viruses. Multidisciplinary Digital Publishing Institute; 2021;13:1773. pmid:34578354
  15. 15. Chandrashekar A, Liu J, Yu J, McMahan K, Tostanoski LH, Jacob-Dolan C, et al. Prior infection with SARS-CoV-2 WA1/2020 partially protects rhesus macaques against reinfection with B.1.1.7 and B.1.351 variants. Sci Transl Med. American Association for the Advancement of Science; 2021;13:eabj2641. pmid:34546094
  16. 16. Brustolin M, Rodon J, Rodríguez de la Concepción ML, Ávila-Nieto C, Cantero G, Pérez M, et al. Protection against reinfection with D614- or G614-SARS-CoV-2 isolates in golden Syrian hamster. Emerg Microbes Infect. Taylor & Francis; 2021;10:797–809. pmid:33825619
  17. 17. Gaudreault NN, Trujillo JD, Carossino M, Meekins DA, Morozov I, Madden DW, et al. SARS-CoV-2 infection, disease and transmission in domestic cats. Emerg Microbes Infect. Taylor & Francis; 2020;9:2322–32. pmid:33028154
  18. 18. Eyre DW, Taylor D, Purver M, Chapman D, Fowler T, Pouwels KB, et al. The impact of SARS-CoV-2 vaccination on Alpha & Delta variant transmission. medRxiv. Cold Spring Harbor Laboratory Press; 2021:2021.09.28.21264260.
  19. 19. Marsh GA, McAuley AJ, Au GG, Riddell S, Layton D, Singanallur NB, et al. ChAdOx1 nCoV-19 (AZD1222) vaccine candidate significantly reduces SARS-CoV-2 shedding in ferrets. NPJ Vaccines. Nature Publishing Group; 2021;6:67–8. pmid:33972565
  20. 20. van Doremalen N, Purushotham JN, Schulz JE, Holbrook MG, Bushmaker T, Carmody A, et al. Intranasal ChAdOx1 nCoV-19/AZD1222 vaccination reduces viral shedding after SARS-CoV-2 D614G challenge in preclinical models. Sci Transl Med. American Association for the Advancement of Science; 2021;13. pmid:34315826
  21. 21. Zhou B, Thao TTN, Hoffmann D, Taddeo A, Ebert N, Labroussaa F, et al. SARS-CoV-2 spike D614G change enhances replication and transmission. Nature. Nature Publishing Group; 2021;592:122–27. pmid:33636719
  22. 22. Ulrich L, Halwe NJ, Taddeo A, Ebert N, Schön J, Devisme C, et al. Enhanced fitness of SARS-CoV-2 variant of concern B.1.1.7, but not B.1.351, in animal models. bioRxiv. Cold Spring Harbor Laboratory; 2021:2021.06.28.450190.
  23. 23. Port JR, Yinda CK, Avanzato VA, Schulz JE, Holbrook MG, van Doremalen N, et al. Increased aerosol transmission for B.1.1.7 (alpha variant) over lineage A variant of SARS-CoV-2. bioRxiv. Cold Spring Harbor Laboratory; 2021:2021.07.26.453518. pmid:34341792
  24. 24. Cool K, Gaudreault NN, Morozov I, Trujillo JD, Meekins DA, McDowell C, et al. Infection and transmission of SARS-CoV-2 and its alpha variant in pregnant white-tailed deer. bioRxiv. Cold Spring Harbor Laboratory; 2021:2021.08.15.456341. pmid:34426811
  25. 25. Rathnasinghe R, Jangra S, Cupic A, Martínez-Romero C, Mulder LCF, Kehrer T, et al. The N501Y mutation in SARS-CoV-2 spike leads to morbidity in obese and aged mice and is neutralized by convalescent and post-vaccination human sera. medRxiv. Cold Spring Harbor Laboratory Press; 2021:2021.01.19.21249592. pmid:33501468
  26. 26. Tarrés-Freixas F, Trinité B, Pons-Grífols A, Romero-Durana M, Riveira-Muñoz E, Ávila-Nieto C, et al. SARS-CoV-2 B.1.351 (beta) variant shows enhanced infectivity in K18-hACE2 transgenic mice and expanded tropism to wildtype mice compared to B.1 variant. bioRxiv. Cold Spring Harbor Laboratory; 2021:2021.08.03.454861.
  27. 27. Montagutelli X, Prot M, Levillayer L, Salazar EB, Jouvion G, Conquet L, et al. The B1.351 and P.1 variants extend SARS-CoV-2 host range to mice. bioRxiv. Cold Spring Harbor Laboratory; 2021:2021.03.18.436013.
  28. 28. Wang R, Zhang Q, Ge J, Ren W, Zhang R, Lan J, et al. Analysis of SARS-CoV-2 variant mutations reveals neutralization escape mechanisms and the ability to use ACE2 receptors from additional species. Immunity. 2021;54:1611–21.e5. pmid:34166623
  29. 29. Pizzorno A, Padey B, Dubois J, Julien T, Traversier A, Dulière V, et al. In vitro evaluation of antiviral activity of single and combined repurposable drugs against SARS-CoV-2. Antiviral Res. 2020;181:104878. pmid:32679055
  30. 30. Funnell SGP, Dowling WE, Munoz-Fontela C, Gsell P-S, Ingber DE, Hamilton GA, et al. Emerging preclinical evidence does not support broad use of hydroxychloroquine in COVID-19 patients. Nat Commun. Nature Publishing Group; 2020;11:4253–4. pmid:32848158
  31. 31. Munster VJ, Flagg M, Singh M, Williamson BN, Feldmann F, Pérez-Pérez L, et al. Subtle differences in the pathogenicity of SARS-CoV-2 variants of concern B.1.1.7 and B.1.351 in rhesus macaques. bioRxiv. Cold Spring Harbor Laboratory; 2021:2021.05.07.443115. pmid:34382034
  32. 32. Rosenke K, Feldmann F, Okumura A, Hansen F, Tang-Huau T, Meade-White K, et al. UK B.1.1.7 variant exhibits increased respiratory replication and shedding in nonhuman primates. bioRxiv. Cold Spring Harbor Laboratory; 2021:2021.06.11.448134. pmid:34159332
  33. 33. Thorne LG, Bouhaddou M, Reuschl A-K, Zuliani-Alvarez L, Ben Polacco , Pelin A, et al. Evolution of enhanced innate immune evasion by the SARS-CoV-2 B.1.1.7 UK variant. bioRxiv. Cold Spring Harbor Laboratory; 2021:2021.06.06.446826. pmid:34127972
  34. 34. Escalera A, Gonzalez-Reiche AS, Aslam S, Mena I, Pearl RL, Laporte M, et al. SARS-CoV-2 variants of concern have acquired mutations associated with an increased spike cleavage. bioRxiv. Cold Spring Harbor Laboratory; 2021:2021.08.05.455290.
  35. 35. Peacock TP, Sheppard CM, Brown JC, Goonawardane N, Zhou J, Whiteley M, et al. The SARS-CoV-2 variants associated with infections in India, B.1.617, show enhanced spike cleavage by furin. bioRxiv. Cold Spring Harbor Laboratory; 2021:2021.05.28.446163.
  36. 36. Thi Nhu Thao T, Labroussaa F, Ebert N, V’kovski P, Stalder H, Portmann J, et al. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform. Nature. Nature Publishing Group; 2020;582:561–65. pmid:32365353
  37. 37. Kim HW, Canchola JG, Brandt CD, Pyles G, Chanock RM, Jensen K, et al. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am J Epidemiol. 1969;89:422–34. pmid:4305198
  38. 38. Rød T, Haug KW, Ulstrup JC. Atypical measles after vaccination with killed vaccine. Scand J Infect Dis. Taylor & Francis; 1970;2:161–65. pmid:25607573
  39. 39. Roy SK, Bhattacharjee S. Dengue virus: epidemiology, biology, and disease aetiology. Can J Microbiol. NRC Research Press 1840 Woodward Drive, Suite 1, Ottawa, ON K2C 0P7; 2021:1–16. pmid:34171205
  40. 40. Wan Y, Shang J, Sun S, Tai W, Chen J, Geng Q, et al. Molecular Mechanism for Antibody-Dependent Enhancement of Coronavirus Entry. J Virol. 2020:94. pmid:31826992
  41. 41. Bolles M, Deming D, Long K, Agnihothram S, Whitmore A, Ferris M, et al. A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge. J Virol. 2011;85:12201–15. pmid:21937658
  42. 42. Tseng C-T, Sbrana E, Iwata-Yoshikawa N, Newman PC, Garron T, Atmar RL, et al. Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus. Poehlmann S, editor. PLoS ONE. Public Library of Science; 2012;7:e35421. pmid:22536382
  43. 43. Weingartl H, Czub M, Czub S, Neufeld J, Marszal P, Gren J, et al. Immunization with modified vaccinia virus Ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets. J Virol. 2004;78:12672–6. pmid:15507655
  44. 44. Munoz FM, Cramer JP, Dekker CL, Dudley MZ, Graham BS, Gurwith M, et al. Vaccine-associated enhanced disease: Case definition and guidelines for data collection, analysis, and presentation of immunization safety data. Vaccine. 2021;39:3053–66. pmid:33637387
  45. 45. Bewley KR, Gooch K, Thomas KM, Longet S, Wiblin N, Hunter L, et al. Immunological and pathological outcomes of SARS-CoV-2 challenge following formalin-inactivated vaccine in ferrets and rhesus macaques. Sci Adv. American Association for the Advancement of Science; 2021;7:eabg7996. pmid:34516768
  46. 46. Hoffmann D, Corleis B, Rauch S, Roth N, Mühe J, Halwe NJ, et al. CVnCoV and CV2CoV protect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2. Nat Commun. Nat Publ Group. 2021;12:4048–7. pmid:34193869
  47. 47. He Y, Qu J, Wei L, Liao S, Zheng N, Liu Y, et al. Generation and Effect Testing of a SARS-CoV-2 RBD-Targeted Polyclonal Therapeutic Antibody Based on a 2-D Airway Organoid Screening System. Front Immunol Frontiers. 2021;12:689065. pmid:34733269
  48. 48. Su S-C, Yang T-J, Yu P-Y, Liang K-H, Chen W-Y, Yang C-W, et al. Structure-guided antibody cocktail for prevention and treatment of COVID-19. PLoS Pathog. 2021;17:e1009704. pmid:34673836
  49. 49. Maisonnasse P, Aldon Y, Marc A, Marlin R, Dereuddre-Bosquet N, Kuzmina NA, et al. COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models. Nat Commun. Nature Publishing Group. 2021;12:6097–10. pmid:34671037
  50. 50. Fenwick C, Turelli P, Perez L, Pellaton C, Esteves-Leuenberger L, Farina A, et al. A highly potent antibody effective against SARS-CoV-2 variants of concern. Cell Rep. 2021;37:109814. pmid:34599871
  51. 51. Haga K, Takai-Todaka R, Matsumura Y, Song C, Takano T, Tojo T, et al. Nasal delivery of single-domain antibody improves symptoms of SARS-CoV-2 infection in an animal model. PLoS Pathog. Public Library of Science; 2021;17:e1009542. pmid:34648602
  52. 52. Huo J, Mikolajek H, Le Bas A, Clark JJ, Sharma P, Kipar A, et al. A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19. Nat Commun. Nature Publishing Group; 2021;12:5469–18. pmid:34552091
  53. 53. Kaplonek P, Wang C, Bartsch Y, Fischinger S, Gorman MJ, Bowman K, et al. Early cross-coronavirus reactive signatures of humoral immunity against COVID-19. Sci Immunol. 2021;6:eabj2901. pmid:34652962
  54. 54. Yamin R, Jones AT, Hoffmann H-H, Schäfer A, Kao KS, Francis RL, et al. Fc-engineered antibody therapeutics with improved anti-SARS-CoV-2 efficacy. Nature. Nature Publishing Group; 2021:1–6. pmid:34547765
  55. 55. Williamson BN, Feldmann F, Schwarz B, Meade-White K, Porter DP, Schulz J, et al. Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2. Nature. Nature Publishing Group; 2020:1–7. pmid:32516797
  56. 56. Kaptein SJF, Jacobs S, Langendries L, Seldeslachts L, Horst ter S, Liesenborghs L, et al. Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity. Proc Natl Acad Sci U S A. National Academy of Sciences; 2020;117:26955–65. pmid:33037151
  57. 57. Rosenke K, Hansen F, Schwarz B, Feldmann F, Haddock E, Rosenke R, et al. Orally delivered MK-4482 inhibits SARS-CoV-2 replication in the Syrian hamster model. Nat Commun. Nature Publishing Group; 2021;12:1–8. pmid:33397941
  58. 58. Cox RM, Wolf JD, Plemper RK. Therapeutically administered ribonucleoside analogue MK-4482/EIDD-2801 blocks SARS-CoV-2 transmission in ferrets. Nat Microbiol. Nature Publishing Group; 2021;6:11–18. pmid:33273742
  59. 59. Boras B, Jones RM, Anson BJ, Arenson D, Aschenbrenner L, Bakowski MA, et al. Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19. Nat Commun. Nature Publishing Group; 2021;12:6055–17. pmid:34663813
  60. 60. de Melo GD, Lazarini F, Levallois S, Hautefort C, Michel V, Larrous F, et al. COVID-19-related anosmia is associated with viral persistence and inflammation in human olfactory epithelium and brain infection in hamsters. Sci Transl Med. American Association for the Advancement of Science; 2021;13. pmid:33941622
  61. 61. Allnoch L, Beythien G, Leitzen E, Becker K, Kaup F-J, Stanelle-Bertram S, et al. Vascular Inflammation Is Associated with Loss of Aquaporin 1 Expression on Endothelial Cells and Increased Fluid Leakage in SARS-CoV-2 Infected Golden Syrian Hamsters. Viruses. Multidisciplinary Digital Publishing Institute; 2021;13:639. pmid:33918079
  62. 62. Becker K, Beythien G, de Buhr N, Stanelle-Bertram S, Tuku B, Kouassi NM, et al. Vasculitis and Neutrophil Extracellular Traps in Lungs of Golden Syrian Hamsters With SARS-CoV-2. Front Immunol. Frontiers. 2021;12:640842. pmid:33912167
  63. 63. Dhakal S, Ruiz-Bedoya CA, Zhou R, Creisher PS, Villano JS, Littlefield K, et al. Sex Differences in Lung Imaging and SARS-CoV-2 Antibody Responses in a COVID-19 Golden Syrian Hamster Model. mBio. 2021;12:e0097421. pmid:34253053
  64. 64. Yuan L, Zhu H, Zhou M, Ma J, Chen R, Chen Y, et al. Gender associates with both susceptibility to infection and pathogenesis of SARS-CoV-2 in Syrian hamster. Signal Transduct Target Ther. Nature Publishing Group; 2021;6:136–8. pmid:33790236
  65. 65. Takahashi T, Ellingson MK, Wong P, Israelow B, Lucas C, Klein J, et al. Sex differences in immune responses that underlie COVID-19 disease outcomes. Nature. Nature Publishing Group; 2020;588:315–20. pmid:32846427
  66. 66. Osterrieder N, Bertzbach LD, Dietert K, Abdelgawad A, Vladimirova D, Kunec D, et al. Age-Dependent Progression of SARS-CoV-2 Infection in Syrian Hamsters. Viruses. Multidisciplinary Digital Publishing Institute; 2020;12:779. pmid:32698441
  67. 67. Gerhards NM, Cornelissen JBWJ, van Keulen LJM, Harders-Westerveen J, Vloet R, Smid B, et al. Predictive Value of Precision-Cut Lung Slices for the Susceptibility of Three Animal Species for SARS-CoV-2 and Validation in a Refined Hamster Model. Pathogens. Multidisciplinary Digital Publishing Institute; 2021;10:824. pmid:34209230
  68. 68. Imai M, Iwatsuki-Horimoto K, Hatta M, Loeber S, Halfmann PJ, Nakajima N, et al. Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development. Proc Natl Acad Sci U S A. National Academy of Sciences; 2020;117:16587–95. pmid:32571934
  69. 69. Salguero FJ, White AD, Slack GS, Fotheringham SA, Bewley KR, Gooch KE, et al. Comparison of rhesus and cynomolgus macaques as an infection model for COVID-19. Nat Commun. Nature Publishing Group; 2021;12:1260–14. pmid:33627662
  70. 70. Ishigaki H, Nakayama M, Kitagawa Y, Nguyen CT, Hayashi K, Shiohara M, et al. Neutralizing antibody-dependent and -independent immune responses against SARS-CoV-2 in cynomolgus macaques. Virology. 2021;554:97–105. pmid:33412411
  71. 71. Rockx B, Kuiken T, Herfst S, Bestebroer T, Lamers MM, Oude Munnink BB, et al. Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model. Science. American Association for the Advancement of Science; 2020;368:1012–15. pmid:32303590
  72. 72. Munster VJ, Feldmann F, Williamson BN, van Doremalen N, Pérez-Pérez L, Schulz J, et al. Respiratory disease in rhesus macaques inoculated with SARS-CoV-2. Nature. 2020;579:265. pmid:32015508
  73. 73. Leist SR, Dinnon KH, Schäfer A, Tse LV, Okuda K, Hou YJ, et al. A Mouse-Adapted SARS-CoV-2 Induces Acute Lung Injury and Mortality in Standard Laboratory Mice. Cell. 2020;183:1070–85.e12. pmid:33031744
  74. 74. Huang K, Zhang Y, Hui X, Zhao Y, Gong W, Wang T, et al. Q493K and Q498H substitutions in Spike promote adaptation of SARS-CoV-2 in mice. EBioMedicine. 2021;67:103381. pmid:33993052
  75. 75. Winkler ES, Bailey AL, Kafai NM, Nair S, McCune BT, Yu J, et al. SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function. Nat Immunol. Nature Publishing Group; 2020;21:1327–35. pmid:32839612
  76. 76. Zheng J, Wong L-YR, Li K, Verma AK, Ortiz ME, Wohlford-Lenane C, et al. COVID-19 treatments and pathogenesis including anosmia in K18-hACE2 mice. Nature. Nature Publishing Group; 2021;589:603–7. pmid:33166988
  77. 77. Frieman MB, Chen J, Morrison TE, Whitmore A, Funkhouser W, Ward JM, et al. SARS-CoV pathogenesis is regulated by a STAT1 dependent but a type I, II and III interferon receptor independent mechanism. PLoS Pathog. Public Library of Science; 2010;6:e1000849. pmid:20386712
  78. 78. Vijay R, Hua X, Meyerholz DK, Miki Y, Yamamoto K, Gelb M, et al. Critical role of phospholipase A2 group IID in age-related susceptibility to severe acute respiratory syndrome-CoV infection. J Exp Med. 2015;212:1851–68. pmid:26392224
  79. 79. Bajaj V, Gadi N, Spihlman AP, Wu SC, Choi CH, Moulton VR. Aging, Immunity, and COVID-19: How Age Influences the Host Immune Response to Coronavirus Infections? Front Physiol. Frontiers. 2020;11:571416. pmid:33510644
  80. 80. Kim Y-I, Yu K-M, Koh J-Y, Kim E-H, Kim S-M, Kim EJ, et al. Age-dependent pathogenic characteristics of SARS-CoV-2 infection in ferrets. Res Sq. 2021. pmid:33821260
  81. 81. Giamarellos-Bourboulis EJ, Netea MG, Rovina N, Akinosoglou K, Antoniadou A, Antonakos N, et al. Complex Immune Dysregulation in COVID-19 Patients with Severe Respiratory Failure. Cell Host Microbe. 2020;27:992–1000.e3. pmid:32320677
  82. 82. Rydyznski Moderbacher C, Ramirez SI, Dan JM, Grifoni A, Hastie KM, Weiskopf D, et al. Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity. Cell. 2020;183:996–1012.e19. pmid:33010815
  83. 83. Singh DK, Singh B, Ganatra SR, Gazi M, Cole J, Thippeshappa R, et al. Responses to acute infection with SARS-CoV-2 in the lungs of rhesus macaques, baboons and marmosets. Nat Microbiol. Nature Publishing Group; 2021;6:73–86. pmid:33340034
  84. 84. Blair RV, Vaccari M, Doyle-Meyers LA, Roy CJ, Russell-Lodrigue K, Fahlberg M, et al. Acute Respiratory Distress in Aged, SARS-CoV-2-Infected African Green Monkeys but Not Rhesus Macaques. Am J Pathol. 2021;191:274–82. pmid:33171111
  85. 85. Speranza E, Purushotham JN, Port JR, Schwarz B, Flagg M, Williamson BN, et al. Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques. bioRxiv. Cold Spring Harbor Laboratory; 2021:2021.09.08.459430.
  86. 86. Ma Y, Lu D, Bao L, Qu Y, Liu J, Qi X, et al. SARS-CoV-2 infection aggravates chronic comorbidities of cardiovascular diseases and diabetes in mice. Animal Model Exp Med. John Wiley & Sons, Ltd; 2021;4:2–15. pmid:33738432
  87. 87. Nishiga M, Wang DW, Han Y, Lewis DB, Wu JC. COVID-19 and cardiovascular disease: from basic mechanisms to clinical perspectives. Nat Rev Cardiol. Nature Publishing Group; 2020;17:543–58. pmid:32690910
  88. 88. Port JR, Adney DR, Schwarz B, Schulz JE, Sturdevant DE, Smith BJ, et al. Western diet increases COVID-19 disease severity in the Syrian hamster. bioRxiv. Cold Spring Harbor Laboratory; 2021:2021.06.17.448814. pmid:34159329
  89. 89. Kyriakidis NC, López-Cortés A, González EV, Grimaldos AB, Prado EO. SARS-CoV-2 vaccines strategies: a comprehensive review of phase 3 candidates. NPJ Vaccines. Nature Publishing Group; 2021;6:28–17. pmid:33619260
  90. 90. Mahase E. Covid vaccine could be rolled out to children by autumn. BMJ. British Medical Journal Publishing Group; 2021;372:n723. pmid:33727232
  91. 91. McMahan K, Yu J, Mercado NB, Loos C, Tostanoski LH, Chandrashekar A, et al. Correlates of protection against SARS-CoV-2 in rhesus macaques. Nature. Nature Publishing Group; 2021;590:630–34. pmid:33276369