key: cord-1026073-m9ynza4u
authors: Lee, Pureum; Kim, Doo-Jin
title: Newly Emerging Human Coronaviruses: Animal Models and Vaccine Research for SARS, MERS, and COVID-19
date: 2020-07-22
journal: Immune Netw
DOI: 10.4110/in.2020.20.e28
sha: 0752b2bb3551a6c54532ad34b0810a6d8b6cbed9
doc_id: 1026073
cord_uid: m9ynza4u

The recent emergence of the novel coronavirus (CoV) or severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a global threat to human health and economy. As of June 26, 2020, over 9.4 million cases of infection, including 482,730 deaths, had been confirmed across 216 countries. To combat a devastating virus pandemic, numerous studies on vaccine development are urgently being accelerated. In this review article, we take a brief look at the characteristics of SARS-CoV-2 in comparison to SARS and Middle East respiratory syndrome (MERS)-CoVs and discuss recent approaches to coronavirus disease-2019 (COVID-19) vaccine development.

Coronaviruses are positive-sense RNA viruses belonging to the family Coronaviridae. They are divided into 4 genera: alpha (α), beta (β), gamma (γ), and delta (δ) coronaviruses, based on their phylogenetic relationships and genomic structures. αand β-coronaviruses infect only mammals whereas the γand δ-coronaviruses mainly infect birds (1) . Typically, coronaviruses are known to cause only mild illnesses, like the common cold in humans, but the outbreak of severe acute respiratory syndrome (SARS) in 2002 (2) demonstrated that coronaviruses originating from other animal species may cross the species barrier and could become life-threatening pathogens in humans. A decade after the SARS outbreak, Middle East respiratory syndrome coronavirus (MERS-CoV), another pathogenic coronavirus, emerged in Saudi Arabia (3). Most recently, another β-coronavirus-severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-has been newly identified from a cluster of patients with severe pneumonia (4,5). To date, 2 α-coronaviruses (human coronavirus [HCoV]-229E and HCoV-NL63) and 5 β-coronaviruses (HCoV-OC43, HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2) have been identified that infect humans (1) . clinical symptoms of SARS are similar to those of other respiratory infections, like influenza. During the initial phase of infection, patients with SARS exhibited fever, cough, sore throat, and other mild symptoms, and some subsequently progressed to severe pneumonia (11) . High levels of pro-inflammatory cytokines and chemokines were detected in the sera of SARS patients with severe disease (12) . They also displayed low levels of anti-inflammatory cytokines, such as IL-10, compared to those of patients with mild symptoms.

The receptor binding domain (RBD) of S protein binds to host angiotensin-converting enzyme 2 (ACE2) for their entry into cells (13) . ACE2 is expressed on a wide variety of body tissues and cells, including small intestine epithelium, arterial and venous endothelium, arterial smooth muscle, respiratory tract epithelium, alveolar monocytes, and alveolar macrophages (14) . Owing to the widespread expression of ACE2 throughout the body, SARS-CoV infects various tissues and causes lesions. Specifically, SARS-CoV primarily infects airway epithelial cells, resulting in the induction of large amounts of chemokines, such as CCL2, CCL3, CCL5, and CXCL10. The virus also infects hematopoietic cells, like dendritic cells (DCs) and macrophages. In such cases, DCs and macrophages exhibit the upregulated expression of pro-inflammatory cytokines and chemokines, such as TNF-α, IL-6, and CXCL10, but downregulated or delayed type I IFN (IFN-α/β) response (15) . Consequently, the excess concentrations of pro-inflammatory molecules recruit various inflammatory immune cells into the lungs, leading to consolidation, hemorrhages, edema, and diffuse alveolar damage (DAD) (16) . (18), which was the most unique and largest MERS outbreak outside the Arabian Peninsula.

Major clinical symptoms of MERS are fever, non-productive cough, dyspnea, myalgia, and sore throat (19) . Unlike patients with influenza and SARS, certain patients with MERS distinctively presented gastrointestinal symptoms, including diarrhea and vomiting (20) . The majority of MERS patients progressed to severe pneumonia; particularly, immunocompromised individuals and patients with comorbidities exhibited high incidences of acute respiratory distress syndrome (ARDS) and renal dysfunction (21) . Similar to SARS, in MERS patients, a dysregulated immune response is thought to be the cause of pathological changes, such as extensive infiltration of immune cells into the lungs (22) .

In contrast to the case of SARS, MERS-CoV particles or MERS-CoV-specific Abs were detected in a large number of dromedary camels in the Middle East and North Africa (23). This strongly indicates that the virus, which is thought to originate from bats (24,25), has been circulating for more than several decades in dromedary camels in those areas, which may be a reason for continuing zoonotic transmission of MERS-CoV. Human-to-human transmission of MERS-CoV mainly occurs through the nosocomial route, especially between hospitalized patients (26), probably because virus shedding takes place efficiently after the onset of disease symptoms.

DPP4 is expressed in the respiratory tract epithelium, kidney, small intestine, liver, prostate, and also on activated leukocytes (28). The virus primarily infects airway epithelial cells, resulting in delayed IFN responses and upregulated pro-inflammatory cytokines, such as IL-6, IL-1β, and IL-8 (29). MERS-CoV-infected macrophages and DCs also produce high levels of pro-inflammatory cytokines and chemokines, such as CCL2, CCL3, CCL5, and IL-2 (30,31), and the concentration of these soluble factors closely correlates with disease severity (32). Increased numbers of neutrophils and monocytes were observed in the lungs of these types of patients (22) , indicating that these cells are responsible for lung immunopathology. Similar to SARS-CoV, the S protein of SARS-CoV-2 binds to ACE2 as its receptor (38,39), and subsequently trigger fusion of viral and cellular membranes, thereafter entering host cells. As such, amino acid sequence and distribution of ACE2 is the major determinant of host and cell tropism (40). In the human body, ACE2 is expressed at a high level in the small intestine, testis, and kidneys, and at a relatively low level in the lungs and heart in healthy individuals. However, in the case of smokers and patients with heart conditions, ACE2 levels are elevated compared to that in the healthy (41,42), partially accounting for the high pathogenicity in those populations.

Most patients with COVID-19 exhibit mild to moderate clinical symptoms, such as fever and dry cough, and sometimes muscle and/or joint pain (43). However, the elderly or individuals with underlying diseases, such as asthma, heart conditions, and diabetes, are more vulnerable to SARS-CoV-2, leading to severe pneumonia or ARDS, the main cause of COVID-19-related death (43). Other less common symptoms have also been reported, such as gastrointestinal symptoms (44) and loss of taste or smell (45).

Upon infection, SARS-CoV-2 activates the innate immune system and induces proinflammatory cytokines and chemokines in the lungs along with recruitment of monocytes and T cells (43). In most healthy individuals, this local immune response contributes to the clearance of viral infection, but in patients with preconditions, dysregulated immune response results in massive infiltration of immune cells, respiratory failure, or systemic inflammation. In particular, unlike other respiratory viruses causing mild symptoms, SARS-CoV-2 is unique, as it drives low type I and III interferon levels and a moderate IFN-stimulated gene (ISG) response (46,47). Consistent with these observations, SARS-CoV-2 ORF3b, a 22-amino acid-long nonstructural protein, exhibited a significantly stronger antagonistic activity against type I IFN induction than that displayed by SARS-CoV ORF3b ortholog (48). Contrary to this weak antiviral response, inflammatory response represented by the production of proinflammatory cytokines and chemokines, such as IL-6, CCL8, and CXCL9, was markedly elevated under SARS-CoV-2 infection both in vitro and in vivo (46). Patients with severe COVID-19 also exhibited abundant distribution of proinflammatory monocytederived macrophages in the bronchoalveolar lavage fluid (BALF) (49) and high serum levels of proinflammatory cytokines and chemokines, such as IL-1β, IL-2, IL-6, IL-8, IL-17, IFN-γinduced protein (IP)-10, MCP-1, TNF-α, G-CSF, and GM-CSF (43,50,51). This imbalancedlow antiviral but high inflammatory-host response is believed to be a major factor affecting disease outcome.

In addition to uncontrolled innate immune responses, impaired adaptive immune responses can affect disease severity. The number of lymphocytes including T, B, or NK cells was significantly reduced in patients with severe COVID-19 requiring intensive care unit (ICU) care (50-52). Decreased T cell number has also been observed in patients with SARS (53), and MERS-CoV has been reported to infect human T lymphocytes and activate apoptotic pathways in the infected cells (54). Further investigation is required to elucidate the reason underlying the decrease in the number of lymphocytes in patients with severe COVID-19. It is intriguing that T cells from patients with COVID-19 highly express PD-1 and T-cell immunoglobulin mucin-3 (TIM-3), which are exhaustion markers (52). Additionally, the frequency of T and NK cells producing CD107a, IFN-γ, IL-2, and granzyme B was significantly reduced in patients with severe infection, compared to those from healthy controls (55). However, despite the increased exhaustion marker levels and decreased cellular function, it should be carefully defined whether the T cells are really "exhausted" by continuous antigenic stimulation through Ag-specific TCRs.

To elucidate the mechanisms of viral pathogenesis and develop optimal prophylactic and therapeutic strategies for newly emerging human coronaviruses, several animal models have been developed and evaluated ( Table 1) .

As mentioned earlier, SARS-CoV and SARS-CoV-2 enter target cells by binding to ACE2 as their receptor (38-40). In mice, SARS-CoV is able to interact with murine ACE2 and replicate in mouse tissues, including the lungs and small intestine (57), but disease symptoms are limited to mild respiratory disease and minimal bodyweight loss that is less than 5%. Aged mice present a relatively larger number of severe symptoms than young mice (59) (60) (61) . To improve the availability of a murine SARS model, mouse-adapted SARS-CoV strains (149, 150) and transgenic mice expressing human ACE2 (hACE2) were developed (62, 63) . Myeloid differentiation primary response 88 (MyD88) as well as STAT1 knock-out mice presented severe respiratory diseases, like pneumonitis and bronchiolitis, along with reduced survival compared to wild-type mice (58, 64, 65) , suggesting that innate immunity involved with these molecules plays an important role in the clearance of SARS-CoV.

In addition to mice, various other animal models are available for SARS-CoV studies. Golden Syrian and Chinese hamsters (66) and ferrets (67, 68) are susceptible to SARS-CoV infection and display moderate to severe respiratory symptoms. SARS-CoV infects nonhuman primates (NHPs), including rhesus macaques, cynomolgus macaques, African green monkeys, common marmosets, squirrel monkeys, and mustached tamarins because these NHP species express a form of ACE2 closely related to that of humans (69-71). More importantly, the virus successfully replicates in these NHPs and causes severe symptoms, like fever, pneumonitis, diarrhea, and hepatitis (72).

As SARS-CoV-2 was revealed to also utilize ACE2 for viral entry (38-40), SARS animal models were promptly tested in SARS-CoV-2 studies. hACE2 transgenic mice exhibited moderate interstitial pneumonia (84) , and Golden Syrian hamsters presented clinical symptoms and histopathological findings closely resembling what is observed in humans (85) . In ferrets, SARS-CoV-2 viral RNA was detected in the nasal turbinate, soft palate, and tonsil, but the viral infection induced only mild clinical symptoms (86) . In NHP models, the virus was excreted from the respiratory tract and detected in multiple organs in virusinfected cynomolgus macaques, but they did not develop any clinical signs (88). Aged rhesus macaques exhibited more severe and diffuse pneumonia along with serious inflammatory responses versus young monkeys (89), suggesting the age is a decisive factor in both NHP models and humans. SARS-CoV-2 susceptibility was also investigated in domesticated animals-SARS-CoV-2 can efficiently replicate in cats but does so poorly in dogs, pigs, chickens, and ducks (87). Most recently, a new mouse model using recombinant adenovirus 5 expressing hACE2 (Ad5-hACE2) was reported (90), which is similar to the model developed for a MERS study by the same group (73). Upon intranasal transduction with Ad5-hACE2, mice transiently expressed hACE2 in their respiratory tract and exhibited significant viral replication and lung inflammation upon subsequent SARS-CoV-2 infection.

MERS-CoV employs host cellular DPP4 as its receptor for entry (27). Whereas humans and NHPs are susceptible to MERS-CoV infection, hamsters, ferrets, and mice are not because of differences in major amino acid sequences of DPP4 (151) (152) (153) . The first model for MERS-CoV study was based on mice transduced with recombinant adenovirus expressing human DPP4 (Ad5-hDPP4) (73). The Ad5-hDPP4-transduced mice developed clinical symptoms including pneumonitis and lung edema upon MERS-CoV infection. Global or tissue-specific hDPP4 transgenic mice were successfully infected with MERS-CoV and displayed respiratory symptoms and weight loss (74, 75) . hDPP4 knock-in mice have also been developed within which murine DPP4 is replaced by hDPP4 using CRISPR/Cas9 (77, 78) .

In addition to these mouse models, dromedary camels, an intermediate animal in MERS-CoV transmission, and alpacas were tested for a MERS study. Although they were susceptible to the virus, they were asymptomatic or exhibited, if any, only mild respiratory symptoms (79, 80) . In NHPs, MERS-CoV effectively infected the host cells and replicated within their lungs (81-83), but disease severity was higher in the common marmosets than that in rhesus macaques. Furthermore, symptoms observed in severe patients, such as lung consolidation and liver or kidney failure, were reproduced only in common marmosets (76) , indicating that they are a more reliable animal model for MERS study.

Although there are still no approved vaccines for SARS and MERS, studies on the two previous coronaviruses provided important information about a strategy and considerations for COVID-19 vaccine development. First, as S protein, especially RBD, was known to be responsible for binding to host receptors, it has been extensively evaluated as a primary target Ag for vaccine development. Second, the majority of vaccine studies have utilized various recombinant vaccine platforms, including recombinant proteins, nucleic acids, and virus-vectored vaccines, rather than conventional live-attenuated or inactivated virus platforms ( Fig. 1 and Table 1 ).

As the live-attenuated virus vaccine is composed of almost all proteins of the virus, immune responses induced by attenuated virus vaccination are most similar to those by real viral infection (154) . In a conventional method, live-attenuated virus has been made by serial culture of the virus, which leads to a spontaneous deletion of or mutation within a pathogenic gene.

However, in recent studies, recombination technology-based modification of the target gene has been more widely studied in coronavirus vaccines. Among the coronavirus proteins, E and nsp16 have been thought of as the most potential targets because of their potential association with the virulence in vivo (9). Immunization of engineered mutant SARS-CoV lacking the E protein (rSARS-CoV-ΔE) provided protective immunity in hACE2 transgenic mice and golden Syrian hamsters against viral challenge (91,92). E gene-deleted MERS-CoV (rMERS-CoV-ΔE) was able to replicate only by providing E protein in trans, but unable to propagate in vivo (94). Recombinant MERS-CoV lacking the accessory genes 3, 4a, and 5, was also replicationcompetent in vitro but propagation-defective in vivo, indicating that recombinant MERS-CoV could be a safe and promising vaccine candidate 

Recombinant protein vaccines have long been studied and assessed in terms of efficacy and safety. Many researchers have also evaluated human coronavirus vaccines, mainly focusing on S and RBD proteins of SARS-and MERS-CoVs. Immunization with the full-length, extracellular domain of S proteins or Fc-fused RBD proteins of SARS-CoV induced potent neutralizing Abs in mice and/or rabbits (102-105). Trimeric S or RBD proteins also induced humoral and cellular immune responses and provided protection against SARS-CoV infection in hamsters (106) . Similar to the results from SARS vaccine studies, recombinant S, RBD, Fcfused RBD, and trimeric RBD proteins of MERS-CoV also elicited neutralizing Abs in various animal models, including mice and monkeys, and exhibited protective effects upon viral challenge (107) (108) (109) (110) (111) (112) (113) (114) 116) . Immunization of the N-terminal domain (NTD) of the MERS-CoV S protein also led to protection against MERS-CoV challenge in a transient hDPP4-expressing mice model (Ad5-hDPP4 mice) (115) . In most of these experiments, recombinant protein Ags were used with an adjuvant, such as alum and MF59, to increase Ab or cell-mediated immune responses. Intriguingly, MERS-CoV S proteins alone self-assemble into nanoparticles of a size of approximately 25 nm. With this, the S nanoparticles effectively induce neutralizing Abs and Th1-type cellular immune responses in mice and NHPs (116, 123) , providing protection against MERS-CoV infection in mice (117) . In addition, heterologous prime-boost vaccination with adenoviral vector-expressing S and S nanoparticles led to balanced Th1/Th2 responses and safeguarded mice from MERS-CoV challenge (118) .

In terms of SARS-CoV-2, Novavax (Gaithersburg, MD, USA) is developing a spike nanoparticle vaccine at phase 1/2 clinical stage, and another 50 institutions are working on recombinant protein vaccines focusing on S or RBD proteins at a pre-clinical stage (148) . One of the most outstanding advances in the recent study of recombinant protein vaccines is the design of a prefusion form of viral surface Ags based on structural biology. Target Ags expressed as a stable prefusion form induced more potent and high-affinity neutralizing Ab responses than wild-type proteins in various infectious disease models (159) (160) (161) (162) . Several institutions, including Queensland University (Brisbane, Australia) and Clover Biopharmaceuticals (Chengdu, China), are applying this technology to COVID-19 vaccine development.

Meanwhile, for the optimal efficacy and dose sparing of recombinant protein Ags, it is essential to develop a vaccine in combination with an appropriate adjuvant. In previous SARS and MERS vaccine studies, the effects of diverse adjuvants such as alum (106, 108, 109, 115, 116, 118, 123) , MF59 ® (109, 110, 112) , Matrix™ M (116, 117) , Montanide ISA™ 51 (109, 111, 113) , and monophosphoryl-lipid A plus trehalose dicorynomycolate (MPL ® + TDM) (102,104,114), have been widely tested. During the development of a recombinant protein-based COVID-19 vaccine, the choice of an adjuvant would be a considerable factor affecting the quality of the immune response, the efficacy and safety of the vaccine, and the economic feasibility of the developer.

VLPs are nano-sized particles composed of viral proteins with self-assembly properties. They mimic the morphology of a real virus particle but do not replicate owing to the lack of genomic material. As VLPs maintain the ideal conformation of native Ags, they can elicit an appropriate and strong immune response (163) .

VLPs of SARS-and MERS-CoVs were produced by coexpressing the S, E, and M proteins from insect or mammalian cells (119, 123) , and these VLPs induced potent neutralizing Abs and Th1-biased cellular immune responses in mice and NHPs (120, 121, 123) . Interestingly, SARS-CoV S and influenza virus matrix 1 (M1) coexpression efficiently formed chimeric VLP and induced protective immunity against SARS-CoV (122). At present, ten COVID-19 vaccine candidates are under pre-clinical investigation on the basis of VLP technology (148) .

Since it was first reported that immunization of naked plasmid DNA encoding foreign protein induces an Ag-specific immune response in mammals in the early 1990s (164), DNA vaccines have been widely tested in various pathogen models. Although DNA vaccines have not been approved for humans to date, their immunogenicity and therapeutic effects have long been tested across various clinical trials for infectious diseases as well as human papillomavirusmediated cervical intraepithelial neoplasia (165, 166) . DNA vaccines expressing full-length or truncated forms of SARS-CoV S protein induced humoral and cellular immunity, supplying protection against SARS-CoV infection in murine models (98, 124, 125) . Further, heterologous prime-boost immunization with DNA vaccines and inactivated SARS-CoV induced strong CD4 + T cell and Ab responses (126) . Meanwhile, DNA vaccines encoding SARS-CoV N proteins induced Abs and T cell responses in mice, but their protective efficacy has not been fully investigated (127, 128) . In MERS vaccine studies, immunization of full-length or https://immunenetwork.org S1 subunit-expressing plasmids also induced neutralizing Abs and T cell responses in mice, camels, and NHPs (114, 129, 130) , while also alleviating clinical symptoms upon MERS-CoV infection in a rhesus macaque model (129) . mRNA vaccines are the most recent vaccine technology characterized by rapid development and production along with high potency. Recently, encapsulation and delivery methods have been substantially improved-the use of carrier molecules, such as liposomes, cationic polymers, and polysaccharide particles, significantly increases delivery efficacy, allowing for rapid uptake and high expression of target Ags. Owing to the safety, potent efficacy, as well as mass and prompt producibility, mRNA vaccines are being extensively evaluated in various infectious diseases and cancers (167) . Yet, no striking results have been reported in SARS and MERS vaccine studies. 

As vesicular stomatitis virus (VSV)-based Ebola vaccine (ERVEBO ® ) has been approved for human use by the Food and Drug Administration (FDA) of the USA in 2019 (168) , the use of viral vectors for vaccines against infectious diseases appears to be more flexible than how it was before. Viral vector-based vaccines are able to induce strong and rapid Ab and cellmediated immune responses, and several viral vectors have been developed to date, including VSV, modified vaccinia Ankara (MVA), adenovirus (Ad), and adenovirus-associated virus (AAV) (169) . In coronavirus vaccine studies, Ad and MVA are the most frequently employed. Replication-defective adenoviral vector expressing S and N proteins of SARS-CoV elicited humoral and cellular immune responses in mice (98, 170) . MVA expressing SARS-CoV S protein induced neutralizing Ab responses in mice, ferrets, and NHPs (133) (134) (135) , and reduced lung viral titer in SARS-CoV-challenged mice (135) . Immunization with adenoviral vector encoding S protein of MERS-CoV induced systemic neutralizing Abs and T cell responses (136, 137) . To avoid pre-existing immunity against human adenovirus, chimpanzee adenovirus (ChAdOx1) was utilized in recent vaccine development (138) . The ChAdOx1 vector encoding MERS-CoV S induced neutralizing Abs and T cell responses in hDPP4 transgenic mice (139, 140) , and reduced virus shedding and nasal discharge in dromedary camels upon MERS-CoV infection (141) . MVA encoding the S protein of MERS-CoV also induced neutralizing Abs and T cell responses, protecting Ad-hDPP4-transduced mice and camels from challenge with MERS-CoV (139, 142, 143) . MVA expressing N protein of MERS-CoV elicited CD8 + T cell responses in mice, but its protective efficacy was not determined (144) . In addition to adenovirus and MVA, several viruses, such as Newcastle disease virus, live-attenuated measles virus (MV), rabies virus, Venezuelan equine encephalitis virus, and VSV have also been investigated in the context of MERS vaccine studies ((73, [145] [146] [147] 169) .

A total of 37 vaccine institutions are developing viral vector-based COVID-19 vaccines, and among them, University of Oxford (Oxford, UK) and CanSino Biological (Tianjin, China) are performing phase 1 to 2b/3 clinical trials using a replication-deficient chimpanzee adenovirus (ChAdOx1) and adenovirus type 5 (Ad5), respectively (148) .

Based on the reports introduced above, the Ab response inhibiting the interaction between S or RBD and the corresponding receptors is sufficient for the prevention of SARS-and MERS-CoV infection. Passive transfer of human monoclonal Abs also provided considerable protection against subsequent viral challenge in mice (171) (172) (173) . Taken together, these results suggest that S is a promising target Ag for coronavirus vaccines. Meanwhile, the contribution of T cell immunity for the prevention and clearance of the virus has been widely advocated. CD8 + T cells play a crucial role in viral clearance by secreting effector molecules directly to infected cells (73, 174, 175) . Airway CD4 + T cells also mediate protective immunity against SARS-and MERS-CoV infection through rapid production of IFN-γ (176) . However, the longterm efficacy and safety of SARS or MERS vaccines in humans has not been tested to date. Moreover, in some animal studies, vaccine-induced or monoclonal S-specific neutralizing Abs markedly enhanced the infectivity of SARS-and MERS-CoVs (177, 178) , necessitating further dedicated investigation.

As discussed previously, vaccine technology has significantly advanced over the last several decades. We also have gained useful information and materials for the study of vaccines for the novel coronavirus-how the virus enters the host cells, which Ag we should target, and what kind of animal models we can use. From the aspect of a pre-clinical study, some scientists appear to already have several successful vaccine candidates. However, in terms of a COVID-19 vaccine that is applicable to humans, several factors remain to be considered and intensively investigated.

First, safety issues must be initially evaluated. Certain vaccine formulations have induced sub-optimal Abs and inappropriate Th2-mediated immune responses, leading to Ab dependent enhancement (ADE) and/or vaccine-associated enhanced respiratory disease (VAERD) (99, [179] [180] [181] [182] [183] . Additionally, each candidate should be also tested for toxicity in rats or rabbits. Although nucleic acid vaccines are regarded as safe in the aspect of nonclinical toxicology, long-term safety of an RNA vaccine in humans should be carefully investigated.

The next factor to consider during COVID-19 vaccine development is efficacy, particularly in the elderly and immunocompromised. The mortality of the disease manifests a close correlation with age: less than 1.0% under the age 50, but significantly increasing up to 1.25, 3.99, and 8.61% in the 50s, 60s, and 70s, respectively, and surpassing 13% over 80 (184) . This strongly suggests that the primary target population for COVID-19 vaccine should be the elderly. In the case of conventional vaccines, high-dose or adjuvanted vaccines are recommended to enhance weak immune responses in those populations (185) (186) (187) . Taking this into account, the efficacy of COVID-19 vaccines in the elderly or immunocompromised must be carefully assessed. Nevertheless, even if a COVID-19 vaccine is unsatisfactory in those populations, it might still be beneficial because of indirect protection by establishing "herd immunity".

Other important questions to be addressed are how each immune response contributes to the protection against or clearance of the virus and for how long this effect can last following vaccination or natural infection and recovery. In most pre-clinical vaccine studies, immunization of an S Ag or a part of it efficiently induced a potent neutralizing Ab response. Transfer of a SARS-CoV-2-specific monoclonal Ab and convalescent plasma also provided significant protection against the disease in an animal model and alleviated disease severity in humans (188, 189) . These reports suggest that neutralizing Abs play a key role in the protection or clearance, although partial, of the virus. However, the precise mechanism and extent of contribution of virus-specific T cells to the quality, quantity, and duration of the Ab response have not been addressed, and whether T cells per se provide sufficient protection or exert a therapeutic effect remains unknown (190) . This knowledge is particularly important because it can provide critical information for designing a COVID-19 vaccine and developing a quarantine policy. Currently, the therapeutic effect of adoptively-transferred SARS-CoV-2specific T cells is being tested in a clinical trial (ClinicalTrials.gov: NCT04351659).

This review has presented a brief introduction to three human coronaviruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, and summarized previous and current coronavirus vaccine studies. In addition to the scientific issues discussed herein, there also remain several problems to be resolved in the effort to produce an "available" COVID-19 vaccine-the arrangement of existing infrastructure or build-up of new facilities for mass production, distribution of final goods, and vaccination of large proportions of the population, and so on. Facing a novel coronavirus pandemic, we are engaging in desperate efforts for the development of a safe and effective vaccine. Ultimately, the information in this review will be beneficial and valuable for a better understanding of human coronaviruses and COVID-19 vaccine development.

SARS-CoV-2 vaccines: status report

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Summary of probable SARS cases with onset of illness from 1

Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study

Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome

Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus

Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

Chemokine up-regulation in SARS-coronavirus-infected, monocyte-derived human dendritic cells

Lung pathology of fatal severe acute respiratory syndrome

World Health Organization. MERS situation update

MERS outbreak in Korea: hospital-to-hospital transmission

Clinical course and outcomes of critically ill patients with Middle East respiratory syndrome coronavirus infection

Human intestinal tract serves as an alternative infection route for Middle East respiratory syndrome coronavirus

Middle East respiratory syndrome

Clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of Middle East respiratory syndrome coronavirus infection in the united Arab Emirates

Resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires Stat1

Molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease

Aged BALB/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans

Age-related increases in PGD 2 expression impair respiratory DC migration, resulting in diminished T cell responses upon respiratory virus infection in mice

Mice transgenic for human angiotensin-converting enzyme 2 provide a model for SARS coronavirus infection

Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus

SARS-CoV pathogenesis is regulated by a STAT1 dependent but a type I, II and III interferon receptor independent mechanism

MyD88 is required for protection from lethal infection with a mouse-adapted SARS-CoV

Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters

The SARS-CoV ferret model in an infection-challenge study

Virology: SARS virus infection of cats and ferrets

Macaque model for severe acute respiratory syndrome

Replication of SARS coronavirus administered into the respiratory tract of African Green, rhesus and cynomolgus monkeys

Generation of a transgenic mouse model of Middle East respiratory syndrome coronavirus infection and disease

Middle East respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4

Animal models of Middle East respiratory syndrome coronavirus infection

Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice

A mouse model for MERS coronavirus-induced acute respiratory distress syndrome

Infection, replication, and transmission of Middle East respiratory syndrome coronavirus in alpacas

Replication and shedding of MERS-CoV in upper respiratory tract of inoculated dromedary camels

Pneumonia from human coronavirus in a macaque model

Infection with MERS-CoV causes lethal pneumonia in the common marmoset

An animal model of MERS produced by infection of rhesus macaques with MERS coronavirus

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice

Simulation of the clinical and pathological manifestations of coronavirus disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility

Infection and rapid transmission of SARS-CoV-2 in ferrets

Antibodies against trimeric S glycoprotein protect hamsters against SARS-CoV challenge despite their capacity to mediate FcγRII-dependent entry into B cells in vitro

The receptor binding domain of the new Middle East respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies

Recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against Middle East respiratory syndrome coronavirus challenge

Identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against Middle East respiratory syndrome coronavirus

Optimization of antigen dose for a receptor-binding domain-based subunit vaccine against MERS coronavirus

A truncated receptorbinding domain of MERS-CoV spike protein potently inhibits MERS-CoV infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines

A recombinant receptorbinding domain of MERS-CoV in trimeric form protects human dipeptidyl peptidase 4 (hDPP4) transgenic mice from MERS-CoV infection

Intranasal vaccination with recombinant receptor-binding domain of MERS-CoV spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: Implication for designing novel mucosal MERS vaccines

Evaluation of candidate vaccine approaches for MERS-CoV

The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

Purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice

MERS-CoV spike nanoparticles protect mice from MERS-CoV infection

Heterologous prime-boost vaccination with adenoviral vector and protein nanoparticles induces both Th1 and Th2 responses against Middle East respiratory syndrome coronavirus

Efficient assembly and release of SARS coronavirus-like particles by a heterologous expression system

Effect of mucosal and systemic immunization with virus-like particles of severe acute respiratory syndrome coronavirus in mice

Immune responses against severe acute respiratory syndrome coronavirus induced by virus-like particles in mice

Chimeric severe acute respiratory syndrome coronavirus (SARS-CoV) S glycoprotein and influenza matrix 1 efficiently form virus-like particles (VLPs) that protect mice against challenge with SARS-CoV

MERS-CoV viruslike particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques

A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice

Induction of specific immune responses by severe acute respiratory syndrome coronavirus spike DNA vaccine with or without interleukin-2 immunization using different vaccination routes in mice

Augmentation of immune responses to SARS coronavirus by a combination of DNA and whole killed virus vaccines

Enhanced induction of SARS-CoV nucleocapsid protein-specific immune response using DNA vaccination followed by adenovirus boosting in BALB/c mice

Enhancing immune responses against SARS-CoV nucleocapsid DNA vaccine by co-inoculating interleukin-2 expressing vector in mice

A synthetic consensus anti-spike protein DNA vaccine induces protective immunity against Middle East respiratory syndrome coronavirus in nonhuman primates

Immunogenicity of candidate MERS-CoV DNA vaccines based on the spike protein

Immunogenicity of a DNA vaccine candidate for COVID-19

DNA vaccine protection against SARS-CoV-2 in rhesus macaques

Evaluation of modified vaccinia virus Ankara based recombinant SARS vaccine in ferrets

Recombinant modified vaccinia virus Ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region

Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice

Systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of Middle East respiratory syndrome coronavirus

Immunogenicity of an adenoviral-based Middle East respiratory syndrome coronavirus vaccine in BALB/c mice

A novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity

ChAdOx1 and MVA based vaccine candidates against MERS-CoV elicit neutralising antibodies and cellular immune responses in mice

Protective efficacy of a novel simian adenovirus vaccine against lethal MERS-CoV challenge in a transgenic human DPP4 mouse model

Humoral immunogenicity and efficacy of a single dose of ChAdOx1 MERS vaccine candidate in dromedary camels

Protective efficacy of recombinant modified vaccinia virus Ankara delivering Middle East respiratory syndrome coronavirus spike glycoprotein

An orthopoxvirus-based vaccine reduces virus excretion after MERS-CoV infection in dromedary camels

CD8 + T cells responding to the Middle East respiratory syndrome coronavirus nucleocapsid protein delivered by vaccinia virus MVA in mice

Live-attenuated bivalent measles virus-derived vaccines targeting Middle East respiratory syndrome coronavirus induce robust and multifunctional T cell responses against both viruses in an appropriate mouse model

A highly immunogenic and protective Middle East respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform

One-health: a safe, efficient, dual-use vaccine for humans and animals against Middle East respiratory syndrome coronavirus and rabies virus

World Health Organization. Draft landscape of COVID-19 candidate vaccines

A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice

A new mouseadapted strain of SARS-CoV as a lethal model for evaluating antiviral agents in vitro and in vivo

Host species restriction of Middle East respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4

The Middle East respiratory syndrome coronavirus (MERS-CoV) does not replicate in Syrian hamsters

Mouse dipeptidyl peptidase 4 is not a functional receptor for Middle East respiratory syndrome coronavirus infection

Rationalizing the development of live attenuated virus vaccines

Comparative evaluation of two severe acute respiratory syndrome (SARS) vaccine candidates in mice challenged with SARS coronavirus

SARS vaccine protective in mice

Immunogenicity and protective efficacy in monkeys of purified inactivated Vero-cell SARS vaccine

Immunogenicity, safety, and protective efficacy of an inactivated SARS-associated coronavirus vaccine in rhesus monkeys

A proof of concept for structure-based vaccine design targeting RSV in humans

Stalking influenza by vaccination with pre-fusion headless HA mini-stem

Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus

Immunogenicity of a prefusion HIV-1 envelope trimer in complex with a quaternarystructure-specific antibody

Virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development

Genetic immunization is a simple method for eliciting an immune response

A phase II, prospective, randomized, multicenter, open-label study of GX-188E, an HPV DNA vaccine, in patients with cervical intraepithelial neoplasia 3

DNA vaccines: prime time is now

mRNA vaccines -a new era in vaccinology

Approval letter -Ervebo (Ebloa Zaire Vaccine, Live)

Developments in viral vector-based vaccines

Vaccine Research for Coronaviruses: SARS, MERS, and COVID-19

Intranasal vaccination of recombinant adeno-associated virus encoding receptor-binding domain of severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein induces strong mucosal immune responses and provides long-term protection against SARS-CoV infection

Characterization of a human monoclonal antibody generated from a B-cell specific for a prefusionstabilized spike protein of Middle East respiratory syndrome coronavirus

Towards a solution to MERS: protective human monoclonal antibodies targeting different domains and functions of the MERS-coronavirus spike glycoprotein

Human monoclonal antibodies to SARS-coronavirus inhibit infection by different mechanisms

Virus-specific memory CD8 T cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection

T cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice

Airway memory CD4 + T cells mediate protective immunity against emerging respiratory coronaviruses

Antibody-dependent enhancement of virus infection and disease

Molecular mechanism for antibody-dependent enhancement of coronavirus entry

A role for nonprotective complement-fixing antibodies with low avidity for measles virus in atypical measles

A role for immune complexes in enhanced respiratory syncytial virus disease

Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine

Evaluation of antibody-dependent enhancement of SARS-CoV infection in rhesus macaques immunized with an inactivated SARS-CoV vaccine

Enhanced inflammation in New Zealand white rabbits when MERS-CoV reinfection occurs in the absence of neutralizing antibody

Estimates of the severity of coronavirus disease 2019: a model-based analysis

MF59-adjuvanted influenza vaccine (FLUAD ® ) elicits higher immune responses than a non-adjuvanted influenza vaccine (Fluzone ® ): a randomized, multicenter, Phase III pediatric trial in Mexico

Fluzone ® high-dose influenza vaccine

Immunogenicity of intramuscular MF59-adjuvanted and intradermal administered influenza enhanced vaccines in subjects aged over 60: a literature review

Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model

Effectiveness of convalescent plasma therapy in severe COVID-19 patients

SARS-CoV-2 infection protects against rechallenge in rhesus macaques