Cell and animal models of SARS-CoV-2 pathogenesis and immunity

The spread of the novel virus SARS coronavirus 2 (SARS-CoV-2) was explosive, with cases first identified in December 2019, and >22 million people infected and >775,000 deaths as of August 2020. SARS-CoV-2 can cause severe respiratory disease in humans leading to coronavirus disease 2019 (COVID-19). The development of effective clinical interventions, such as antivirals and vaccines that can limit or even prevent the burden and spread of SARS-CoV-2, is a global health priority. Testing of leading antivirals, monoclonal antibody therapies and vaccines against SARS-CoV-2 will require robust animal and cell models of viral pathogenesis. In this Special Article, we discuss the cell-based and animal models of SARS-CoV-2 infection and pathogenesis that have been described as of August 2020. We also outline the outstanding questions for which researchers can leverage animal and cell-based models to improve our understanding of SARS-CoV-2 pathogenesis and protective immunity. Taken together, the refinement of models of SARS-CoV-2 infection will be critical to guide the development of therapeutics and vaccines against SARS-CoV-2 to end the COVID-19 pandemic

Divergent pathogenetic outcomes in BALB/c mice following Omicron subvariant infection

Following the emergence of B.1.1.529 Omicron, the SARS-CoV-2 virus evolved into a significant number of sublineage variants that possessed numerous mutations throughout the genome, but particularly within the spike glycoprotein (S) gene. For example, the BQ.1.1 and the XBB.1 and XBB.1.5 subvariants contained 34 and 41 mutations in S, respectively. However, these variants elicited largely replication only or mild disease phenotypes in mice. To better model pathogenic outcomes and measure countermeasure performance, we developed mouse adapted versions (BQ.1.1 MA; XBB.1 MA; XBB.1.5 MA) that reflect more pathogenic acute phase pulmonary disease symptoms of SARS-CoV-2, as well as derivative strains expressing nano-luciferase (nLuc) in place of ORF7 (BQ.1.1 nLuc; XBB.1 nLuc; XBB.1.5 nLuc). Amongst the mouse adapted (MA) viruses, a wide range of disease outcomes were observed including mortality, weight loss, lung dysfunction, and tissue viral loads in the lung and nasal turbinates. Intriguingly, XBB.1 MA and XBB.1.5 MA strains, which contained identical mutations throughout except at position F486S/P in S, exhibited divergent disease outcomes in mice (Ao et al., 2023). XBB.1.5 MA infection was associated with significant weight loss and ∼45 % mortality across two independent studies, while XBB.1 MA infected animals suffered from mild weight loss and only 10 % mortality across the same two independent studies. Additionally, the development and use of nanoluciferase expressing strains provided moderate throughput for live virus neutralization assays. The availability of small animal models for the assessment of Omicron VOC disease potential will enable refined capacity to evaluate the efficacy of on market and pre-clinical therapeutics and interventions

Sarbecovirus disease susceptibility is conserved across viral and host models

Coronaviruses have caused three severe epidemics since the start of the 21st century: SARS, MERS and COVID-19. The severity of the ongoing COVID-19 pandemic and increasing likelihood of future coronavirus outbreaks motivates greater understanding of factors leading to severe coronavirus disease. We screened ten strains from the Collaborative Cross mouse genetic reference panel and identified strains CC006/TauUnc (CC006) and CC044/Unc (CC044) as coronavirus-susceptible and resistant, respectively, as indicated by variable weight loss and lung congestion scores four days post-infection. We generated a genetic mapping population of 755 CC006xCC044 F2 mice and exposed the mice to one of three genetically distinct mouse-adapted coronaviruses: clade 1a SARS-CoV MA15 (n=391), clade 1b SARS-CoV-2 MA10 (n=274), and clade 2 HKU3-CoV MA (n=90). Quantitative trait loci (QTL) mapping in SARS-CoV MA15- and SARS-CoV-2 MA10-infected F2 mice identified genetic loci associated with disease severity. Specifically, we identified seven loci associated with variation in outcome following infection with either virus, including one, HrS43, that is present in both groups. Three of these QTL, including HrS43, were also associated with HKU3-CoV MA outcome. HrS43 overlaps with a QTL previously reported by our lab that is associated with SARS-CoV MA15 outcome in CC011xCC074 F2 mice and is also syntenic with a human chromosomal region associated with severe COVID-19 outcomes in humans GWAS. The results reported here provide: (a) additional support for the involvement of this locus in SARS-CoV MA15 infection, (b) the first conclusive evidence that this locus is associated with susceptibility across the Sarbecovirus subgenus, and (c) demonstration of the relevance of mouse models in the study of coronavirus disease susceptibility in humans

Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as a live virus vaccine candidate

Background: Due to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus. Methods: Here, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type format or a membrane-anchored format lacking the polybasic cleavage site. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, the immunogenicity and protective efficacy of these NDV-based vaccines were investigated. Findings: We report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly in mice. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs. Interpretation: The results suggested that the NDV vector expressing either the wild type S or membrane-anchored S without the polybasic cleavage site could be used as live vector vaccine against SARS-CoV-2. Funding: This work is supported by an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS) contract, the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract, philanthropic donations and NIH grants

Prefusion-stabilized SARS-CoV-2 S2-only antigen provides protection against SARS-CoV-2 challenge

Ever-evolving SARS-CoV-2 variants of concern (VOCs) have diminished the effectiveness of therapeutic antibodies and vaccines. Developing a coronavirus vaccine that offers a greater breadth of protection against current and future VOCs would eliminate the need to reformulate COVID-19 vaccines. Here, we rationally engineer the sequence-conserved S2 subunit of the SARS-CoV-2 spike protein and characterize the resulting S2-only antigens. Structural studies demonstrate that the introduction of interprotomer disulfide bonds can lock S2 in prefusion trimers, although the apex samples a continuum of conformations between open and closed states. Immunization with prefusion-stabilized S2 constructs elicits broadly neutralizing responses against several sarbecoviruses and protects female BALB/c mice from mouse-adapted SARS-CoV-2 lethal challenge and partially protects female BALB/c mice from mouse-adapted SARS-CoV lethal challenge. These engineering and immunogenicity results should inform the development of next-generation pan-coronavirus therapeutics and vaccines

Ex vivo and in vivo suppression of SARS-CoV-2 with combinatorial AAV/RNAi expression vectors

Despite rapid development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant modalities to curb the pandemic by directly attacking the virus on a genetic level remain highly desirable and are urgently needed. Here we comprehensively illustrate the capacity of adeno-associated virus (AAV) vectors co-expressing a cocktail of three short hairpin RNAs (shRNAs; RNAi triggers) directed against the SARS-CoV-2 RdRp and N genes as versatile and effective antiviral agents. In cultured monkey cells and human gut organoids, our most potent vector, SAVIOR (SARS virus repressor), suppressed SARS-CoV-2 infection to background levels. Strikingly, in control experiments using single shRNAs, multiple SARS-CoV-2 escape mutants quickly emerged from infected cells within 24–48 h. Importantly, such adverse viral adaptation was fully prevented with the triple-shRNA AAV vector even during long-term cultivation. In addition, AAV-SAVIOR efficiently purged SARS-CoV-2 in a new model of chronically infected human intestinal cells. Finally, intranasal AAV-SAVIOR delivery using an AAV9 capsid moderately diminished viral loads and/or alleviated disease symptoms in hACE2-transgenic or wild-type mice infected with human or mouse SARS-CoV-2 strains, respectively. Our combinatorial and customizable AAV/RNAi vector complements ongoing global efforts to control the coronavirus disease 2019 (COVID-19) pandemic and holds great potential for clinical translation as an original and flexible preventive or therapeutic antiviral measure

Protective efficacy of rhesus adenovirus covid-19 vaccines against mouse-adapted sars-cov-2

The global COVID-19 pandemic has sparked intense interest in the rapid development of vaccines as well as animal models to evaluate vaccine candidates and to define immune correlates of protection. We recently reported a mouse-adapted SARS-CoV-2 virus strain (MA10) with the potential to infect wild-type laboratory mice, driving high levels of viral replication in respiratory tract tissues as well as severe clinical and respiratory symptoms, aspects of COVID-19 disease in humans that are important to capture in model systems. We evaluated the immunogenicity and protective efficacy of novel rhesus adenovirus serotype 52 (RhAd52) vaccines against MA10 challenge in mice. Baseline seroprevalence is lower for rhesus adenovirus vectors than for human or chimpanzee adenovirus vectors, making these vectors attractive candidates for vaccine development. We observed that RhAd52 vaccines elicited robust binding and neutralizing antibody titers, which inversely correlated with viral replication after challenge. These data support the development of RhAd52 vaccines and the use of the MA10 challenge virus to screen novel vaccine candidates and to study the immunologic mechanisms that underscore protection from SARS-CoV-2 challenge in wild-type mice

A newcastle disease virus (NDV) expressing a membrane-anchored spike as a cost-effective inactivated SARS-CoV-2 vaccine

A successful severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine must not only be safe and protective, but must also meet the demand on a global scale at a low cost. Using the current influenza virus vaccine production capacity to manufacture an egg-based inactivated Newcastle disease virus (NDV)/SARS-CoV-2 vaccine would meet that challenge. Here, we report pre-clinical evaluations of an inactivated NDV chimera stably expressing the membrane-anchored form of the spike (NDV-S) as a potent coronavirus disease 2019 (COVID-19) vaccine in mice and hamsters. The inactivated NDV-S vaccine was immunogenic, inducing strong binding and/or neutralizing antibodies in both animal models. More importantly, the inactivated NDV-S vaccine protected animals from SARS-CoV-2 infections. In the presence of an adjuvant, antigen-sparing could be achieved, which would further reduce the cost while maintaining the protective efficacy of the vaccine

Antibody potency, effector function, and combinations in protection and therapy for SARS-CoV-2 infection in vivo

SARS-CoV-2, the causative agent of COVID-19, has been responsible for over 42 million infections and 1 million deaths since its emergence in December 2019. There are few therapeutic options and no approved vaccines. Here, we examine the properties of highly potent human monoclonal antibodies (hu-mAbs) in a Syrian hamster model of SARS-CoV-2 and in a mouse-adapted model of SARS-CoV-2 infection (SARS-CoV-2 MA). Antibody combinations were effective for prevention and in therapy when administered early. However, in vitro antibody neutralization potency did not uniformly correlate with in vivo protection, and some hu-mAbs were more protective in combination in vivo. Analysis of antibody Fc regions revealed that binding to activating Fc receptors contributes to optimal protection against SARS-CoV-2 MA. The data indicate that intact effector function can affect hu-mAb protective activity and that in vivo testing is required to establish optimal hu-mAb combinations for COVID-19 prevention

A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures

Coronaviruses are prone to emergence into new host species most recently evidenced by SARS-CoV-2, the causative agent of the COVID-19 pandemic1. Small animal models that recapitulate SARS-CoV-2 disease are desperately needed to rapidly evaluate medical countermeasures (MCMs)2,3. SARS-CoV-2 cannot infect wildtype laboratory mice due to inefficient interactions between the viral spike (S) protein and the murine ortholog of the human receptor, ACE24. We used reverse genetics5 to remodel the interaction between S and mACE2 resulting in a recombinant virus (SARS-CoV-2 MA) that could utilize mACE2 for entry. SARS-CoV-2 MA replicated in both the upper and lower airways of both young adult and aged BALB/c mice. Importantly, disease was more severe in aged mice, and showed more clinically relevant phenotypes than those seen in HFH4-hACE2 transgenic mice. We then demonstrated the utility of this model through vaccine challenge studies in immune competent mice with native expression of mACE2. Lastly, we show that clinical candidate interferon (IFN) lambda-1a can potently inhibit SARS-CoV-2 replication in primary human airway epithelial cells in vitro, and both prophylactic and therapeutic administration diminished replication in mice. Our mouse-adapted SARS-CoV-2 model demonstrates age-related disease pathogenesis and supports the clinical use of pegylated IFN lambda-1a treatment in human COVID-19 infections