A Thermostable mRNA Vaccine against COVID-19

mRNA vaccines have emerged as a leading contender in the fight against the COVID-19 pandemic. In Canada, as of February 2021, two of the three vaccines approved for emergency-use against COVID-19 are mRNA-based. They include Pfizer-BioNTech’s COVID-19 vaccine BNT162b2, and Moderna’s COVID-19 vaccine mRNA-1273 (Government of Canada, 2021). Unlike traditional vaccines that utilize a weakened pathogen or antigens, mRNA vaccines deliver the mRNA of the antigen of interest to the host cells (Pardi et al., 2018). More specifically, the mRNA encodes for the spike protein or the receptor binding domain of the spike protein from SARS-CoV-2 (L. Jackson et al., 2020; Mulligan et al., 2020).

A novel mRNA-based vaccine candidate was examined in a paper published in Cell by Zhang et al., 2020, entitled, “A Thermostable mRNA Vaccine against COVID-19.” Their vaccine, called ARCoV, encodes for the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 (Zhang et al., 2020). Although the previously mentioned mRNA-based vaccines have been approved for emergency use, their performance has not yet been evaluated in animal models (Zhang et al., 2020). The mechanism and efficacy of mRNA vaccines against SARS-CoV-2 are also currently unclear (Zhang et al., 2020). This paper by Zhang et al. therefore attempts to address this gap in the literature, by examining the in vitro and in vivo efficacy of the newly developed thermostable mRNA vaccine, ARCoV (2020).

This study was conducted by researchers from 5 laboratories in China, including State Key Laboratory of Pathogen and Biosecurity (Academy of Military Medical Sciences), School of Medicine (Tsinghua University), State Key Laboratory of Proteomics (Beijing Institute of Lifeomics), Division of HIV/AIDS and Sex-Transmitted Virus Vaccines (National Institutes for Food and Drug Control), and Suzhou Abogen Biosciences Co., Ltd (Zhang et al., 2020).

This paper by Zhang et al. examines the in vitro and in vivo efficacy of a newly developed thermostable mRNA vaccine, called ARCoV (Zhang et al., 2020).

Figure S1: Graphical abstract displaying the experimental overview (Zhang et al., 2020).

1. Design and encapsulation of mRNA encoding SARS-CoV-2 RBD
2. In vivo delivery of ARCoV mRNA-LNP formulation
3. Humoral immune response in ARCoV-vaccinated mice
4. SARS-CoV-2-specific T-cell immune response in ARCoV-vaccinated mice
5. Protection of ARCoV against SARS-CoV-2 challenge in mice
6. Immune correlate of protection against SARS-CoV-2 in ARCoV-vaccinated mice
7. Immunogenicity of ARCoV in cynomolgus macaques

• BALB/c Mice: This particular mouse strain is easily available and very commonly used in biochemical labs, and now has hundreds of available sub-strains (“MGI - Inbred Strains: BALB,” n.d.). They are white albino inbred mice that have relatively long lifespans, and are used across many disciplines (“MGI - Inbred Strains: BALB,” n.d.). The female mice were used in this particular paper because of their low aggression compared to male mice (Zhang et al., 2020).

• Cynomolgus Monkeys: Macaca fascicularis, also known as Crab-eating macaque, is commonly used in laboratories. Due to their similar physiology to humans, cynomolgus monkeys can share infections with humans so they are frequently used as an animal model for disease modelling and drug evaluation.

African green monkey kidney cell Vero, human cervical carcinoma cell HeLa, human embryonic kidney cell HEK293T/F, and human hepatocarcinoma cell Huh7 were used for transfection experiments and RBD expression. The Vero cell line has previously been used in research in investigating the cause of SARS and was one of the few cell lines that allowed replication of SARS-CoV (Kaye et al., 2006). It is now commonly used in researching SARS-CoV-2 due to its high susceptibility to infection, allowing researchers to propagate and isolate the virus (Matsuyama et al., 2020).

The HeLa cell line is commonly used in research due to its ability to replicate indefinitely. While HeLa cells do not naturally express a high amount of ACE2 proteins, they can be genetically engineered to better express this protein (Jackson, 2020). This is useful in studying the infection cycle of SARS-CoV-2 (Jackson, 2020).

The HEK293T/F cell lines are transformed cells with sheared human adenovirus type 5 DNA (Thermo Fisher Scientific, n.d.). It can be easily transfected and used for expression of recombinant proteins DNA (Thermo Fisher Scientific, n.d.), such as the recombinant receptor binding domain used in this study (Zhang et al., 2020).

The Huh7 cell line is commonly used in SARS-CoV-2 research due to its high ability to propagate the SARS-CoV-2 virus (Mirabelli et al., 2020). Since 43% of patients with COVID-19 develop hepatic dysfunction, this cell line is also useful in studying the tropism and etiology of liver disease (Chu et al., 2020). These cell lines were grown in Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum and penicillin (100 U/ml)-streptomycin (100 μg/ml) (Zhang et al., 2020).

• MASCp6: MASCp6 is a mouse adapted SARS-CoV-2 strain (Zhang et al., 2020). This SARS-CoV-2 strain was originally made by Gu et al. (2020) by serially passaging SARS-CoV-2 into 9-month-old BALB/c mice. They inoculated the mice intranasally then sacrificed them three days after infection (Gu et al., 2020). Their results showed that SARS-CoV-2 RNA could be detected in the mice’ lungs. This procedure was performed 6 additional times (Gu et al., 2020) to achieve the mouse-adapted SARS-CoV-2 strain used in the Zhang et al. (2020) study. This viral strain can easily infect and replicate in the lungs and trachea of BALB/c mice, which makes it useful for studying disease in COVID-19 patients (Gu et al., 2020). Experiments performed with this virus were done in a biosafety level 3 facility (Zhang et al, 2020).

• Vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudovirus: VSV-based SARS-CoV-2 pseudovirus is a lab-made virus that expresses the SARS-CoV-2 spike protein (Nie et al., 2020). Due to the limited number of biosafety level 3 facilities available to perform experiments on SARS-CoV-2, there is a need to create a pseudovirus. The VSV-based pseudovirus was made by cloning the spike genes of SARS-CoV-2 into the pcDNA3.1 expression plasmid to generate the pcDNA3.1.S2 envelope recombinant plasmid (Nie et al., 2020). Afterwards, pcDNA3.1.S2 was inserted into the VSV-based pseudovirus so that it would express the spike protein on its surface (Nie et al., 2020).

mRNA Synthesis

Construction of an artificial mRNA is required for producing the SARS-CoV-2 receptor binding domain inside host cells. The mRNA was produced in vitro using T7 RNA polymerase and a linearized DNA template from the ABOP-028 plasmid (Zhang et al., 2020). This plasmid encodes for the SARS-CoV-2 receptor binding domain, 5’ and 3’ untranslated regions, and a poly-A tail (Zhang et al., 2020). The firefly luciferase-encoding mRNA used in detection experiments was made using the ABOP-010 plasmid instead (Zhang et al., 2020).

mRNA Transfection

Transfection is a technique wherein naked DNA or RNA is introduced into eukaryotic cells. HeLa, Huh7, Vero, and HEK293T cells were plated in 24 wells at 200,000 cells/well (Zhang et al., 2020). Eighteen hours after plating, the cells were transfected with mRNA encoding the receptor binding domain or a control mRNA (2 µg/ml) using Lipofectamine 3000 Transfection Reagent. The medium was later replaced with Opti-MEM I Reduced Serum Medium six hours after transfection. Forty-eight hours after transfection, the supernatant was collected, centrifuged at 1000 x g, and mixed with 5 x SDS loading buffer (non-reducing). The samples were loaded for SDS-PAGE and the receptor binding protein was detected using an anti-SARS-CoV-2 receptor binding domain monoclonal antibody on a Western blot (Zhang et al., 2020).

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is an assay that is used in detecting concentrations of protein in samples. This assay is typically performed on a 96-well plate so that many samples can be measured at once (Horlock, n.d.). There are four main types of ELISAs: direct, indirect, sandwich, and competitive. In a direct ELISA, an antigen is immobilized onto the plate and then an antibody conjugated to an enzyme like horseradish peroxidase or alkaline phosphatase will bind to that antigen (“What is an ELISA?,” n.d.). A substrate is added that will react with the enzyme conjugated to the antibody which will produce a signal that can be detected (“What is an ELISA?,” n.d.). An indirect ELISA is similar to a direct ELISA except it has an extra step of adding an unconjugated antibody before the addition of the conjugated antibody (“What is an ELISA?,” n.d.). This is used to amplify the signal produced after adding the final substrate. A sandwich ELISA is the most commonly used method where an antibody is bound to the plate and then the sample containing the antigen is added (“What is an ELISA?,” n.d.). The plate is washed to remove unbound molecules in the sample and then the conjugated antibody is added. Finally, the substrate is added to produce a signal (“What is an ELISA?,” n.d.). A competitive ELISA is useful in detection of small molecules (“What is an ELISA?,” n.d.). An antibody is bound to the plate then an antigen in the sample and the same antigen conjugated to an enzyme are added. The conjugated and non-conjugated antigen will compete for binding to the antibody (“What is an ELISA?,” n.d.). Once a substrate it added, the signal that is produced will be inversely proportional to the amount of antigen found in the sample (“What is an ELISA?,” n.d.).

In the Zhang et al. (2020) study, ELISA was used to measure the in vitro and in vivo expression of the receptor binding domain. They first coated their 96-well plates with 5 µg/ml of human ACE2 at 4°C and left overnight. The plates were then washed once with PBS and blocked with 5% bovine albumin serum (BSA), then washed again with PBS two times. The plates were incubated with either cell culture media or mouse sera at room temperature for 1 hour before three more washes and incubation with SARS-CoV-2 S rabbit monoclonal antibody. After washing with PBS three more times, plates were given horseradish peroxidase-conjugated goat anti-rabbit IgG-Fc antibody and TMB substrate. Absorbance was measured at 450/620 nm using SpectraMax iD3 (Zhang et al., 2020).

Biolayer Interferometry (BLI)

Biolayer interferometry (BLI) was the binding assay used to measure the binding kinetics of the recombinant receptor binding domain and human ACE2 receptor (Zhang et al., 2020). BLI uses a spectrometer to detect interference patterns of light when reflected from an optical layer and a biolayer containing a protein, such as the ACE2 receptor (Yakimchuk, 2011). When the light from these two layers interacts, an interference pattern is produced (Yakimchuk, 2011). This only occurs when a molecule of interest is bound to the biolayer so unbound molecules will not have an effect on the interference pattern (Creative Biolabs, n.d.). The size of the shift in the interference pattern is proportional to the number of molecules bound to the biolayer (Creative Biolabs, n.d.). In addition, these interactions can be measured in real time with accuracy (Creative Biolabs, n.d.).

Zhang et al. (2020) used Streptavidin (SA) Biosensor from ForteBio to capture 10 µg/ml biotin-ACE2 onto the surface of the SA biosensor. Then 75.6, 30.2, 12.1, 4.84 or 1.94 nM purified receptor binding domain proteins underwent association for 900 s and dissociation for 100 s. The K_D, K_on and K_dis were calculated using Data Analysis Octet (Zhang et al., 2020).

Competitive Inhibition Assay

A competitive inhibition assay is used to test the ability of a protein to bind to a receptor and preventing binding of another protein to the same receptor. This is usually done by adding a fluorescent protein of interest to a 96-well plate containing a receptor and then adding a competing protein (Zhang et al., 2020). The amount of fluorescence produced is proportional to the amount of protein of interest bound to the plate.

In the Zhang et al. (2020) study, Huh7 cells were plated on a 96-well plate at 50,000 cells/well. Then 50 µg/ml of BSA, receptor binding domain, or recombinant receptor binding domain were added for 1 hour at 37°C. 650 TCID₅₀/well of pseudovirus was added and the plates were incubated for 1 hour at 4°C. The cells were washed with DMEM medium and cultured before luciferase was added. GloMax® 96 Microplate Luminometer was used to detect luminescence (Zhang et al., 2020).

Lipid Nanoparticle (LNP) Encapsulation of mRNA

A lipid nanoparticle (LNP) is needed for delivering mRNA into host cells. The study by Zhang et al. (2020) uses a method previously described by Ickenstein and Garidel (2019) to encapsulate their mRNA into LNPs. They first dissolved lipids in ethanol containing an ionizable lipid, 1, 2-distearoyl-sn-glycero3-phosphocholine (DSPC), cholesterol, and PEG-lipid (Zhang et al., 2020). This mixture was then mixed with mRNA in 20 mM citrate buffer (pH 4.0) inside a T-mixer. The LNPs spontaneously formed and encapsulated the mRNA inside. The mRNA-LNPs were then filtered in PBS (pH 7.4) through a tangential-flow filtration membrane and concentrated. To control quality of the samples, particle size, distribution, RNA concentration and encapsulation were tested (Zhang et al., 2020).

Dynamic Light Scattering

Dynamic light scattering is a method used to study the diffusion behaviour of macromolecules in solution (Stetefeld et al., 2016). This is useful in studying the size and size distribution of proteins, nucleic acids, and protein complexes (Stetefeld et al., 2016). Zhang et al. (2020) used a Malvern Zetasizer Nano-ZS to perform this technique. Samples were irradiated with red light (λ = 632.8 nm) and the scattered light was measured. Zetasizer V7.13 software was then used to analyze the results (Zhang et al., 2020).

Pseudovirus-based Neutralization Assay

This test looks at neutralizing ability of antibodies. According to Berthold technologies, cells are seeded on a 96-well plate. A pseudovirus containing luciferase is mixed with serial dilutions of a serum of interest. The luciferase is a fluorescent marker which is only expressed after the pseudovirus enters and infects the cell. The mixture of pseudovirus and diluted serum is applied to the 96-well plate, and fluorescence is measured. Increased luciferase expression, i.e. fluorescence, is observed if more pseudoviruses infect the cells. If effective neutralizing antibodies are present in the serum, they would successfully block entry of the virus into the cells, thereby emitting a weaker fluorescent signal (“Pseudovirus Neutralization Assays in SARS-CoV-2 Research - Berthold Technologies,” n.d.).

The pseudovirus-based neutralization assay in this paper was completed according to the method described in previous research (Nie et al., 2020). In the experiment by Zhang et al. (2020), a human cell line, Huh7, was seeded in 96-well plates at 20,000 cells per well. The plate was then incubated for a full day until it appeared confluent. Serial dilutions of ARCoV-vaccinated mouse serum were completed, and incubated with VSV-based pseudovirus for an hour at a temperature of 37°C. The negative control used in this assay was DMEM. Luciferase was added to each well, and its activity was measured using the GloMax® 96 Microplate Luminometer (Promega). The NT50 was determined to be the dilution where relative light units or RLU’s were 50% of the relative light units of the DMEM control (Zhang et al., 2020).

Plaque Reduction Neutralization Test

This test looks at the activity of neutralizing antibodies. The virus of interest is mixed with serial dilutions of the serum of interest, and applied to cells seeded on a 24-well plate (“Plaque Reduction Neutralization Tests (PRNT),” n.d.). The cells are made stationary by blocking with agarose, the plate is stained with crystal violet, and visualized (“Plaque Reduction Neutralization Tests (PRNT),” n.d.). The viruses that were not neutralized by antibodies would be able to infect plated cells, and would create a plaque forming unit (PFU) which appears as a white spot on the purple plate (“Plaque Reduction Neutralization Tests (PRNT),” n.d.). A high number of PFU would indicate that there are low amounts of neutralizing antibody present in the serum (“Plaque Reduction Neutralization Tests (PRNT),” n.d.)

The plaque reduction neutralization test in this paper was performed as described in a previous paper (Li et al., 2018). In the experiment by Zhang et al. (2020), ARCoV-vaccinated mouse serum was serially diluted and mixed with the SARS-CoV-2 infectious virus in a 1:1 ratio, resulting in a mixture containing 200 plaque forming units per millilitre of virus. This mixture was incubated and then plated on a 24-well plate containing Vero cells. After incubation, the mixture was discarded and the cells were fixed and stained with crystal violet. The number of plaque forming units were then quantified. The PRNT50 was defined as the dilution at which plaques were reduced by 50% compared to the control (Zhang et al., 2020).

Flow Cytometry

Flow cytometry enables you to identify cells based on phenotypic markers. According to Thermo Fisher Scientific, specific T cell types can be identified by selecting cell surface markers specific to the T cells of interest. Cells are stained with fluorescent markers for cell-surface markers of T cells, then passed through a laser. The scattering of light provides information on the presence of specific cell surface markers that are observed in the cells of interest, telling you if they were present or absent in the initial sample. (“How a Flow Cytometer Works | Thermo Fisher Scientific - CA,” n.d.)

In this paper by Zhang et al. (2020), a FACSCalibur flow cytometer was used to determine T cell activity in ARCoV-vaccinated mice. One million mouse splenocytes were stimulated with SARS-CoV-2 RBD peptide pool, and incubated for 2 hours at 37°C with 5% CO₂. Brefeldin A was added to the cells and incubated, then washed with PBS. The splenocytes were then stained with fluorescent conjugated antibodies specific to CD3, CD4, CD8, CD44, and CD62L. Data was collected using FlowJo software (Zhang et al., 2020).

ELISPOT (Enzyme-Linked Immunosorbent Spot)

This assay measures frequency of cytokine-secreting cells at a single-cell level (“ELISpot Assay Principle,” n.d.). Cells are cultured on a surface coated with a specific antibody with/without stimuli, then proteins (e.g. cytokines) secreted by the cells will be captured by those antibodies (“ELISpot Assay Principle,” n.d.). After incubation, cells are removed and secreted molecules are detected with a detection antibody (“ELISpot Assay Principle,” n.d.). By using a substrate with a precipitating product instead of a soluble product, you can see spots on the surface of the assay – each spot observed corresponds to a cytokine-secreting cell (“ELISpot Assay Principle,” n.d.).

Pre-coated ELISPOT kits specific to IFN-γ, TNF-α, IL-2, IL-4, or IL-6 were used in this experiment by Zhang et al. (2020). The manufacture’s protocol called for blocking the plates with RPMI, followed by incubating the plate. ARCoV-vaccinated mice splenocytes were added to the plate at 300,000 cells per well, with concanavalin A used as the positive control, and RPMI used as the negative control. After incubation, a wash step occurred with wash buffer, and then specific anti-mouse antibodies were added to each well. Following another incubation step, substrate solution was added for spot detection by the automated ELISPOT reader, AID ELISPOT. The number of spot-forming cells or SFU’s per million cells was calculated (Zhang et al., 2020).

Immunofluorescence Staining

Immunostaining is a technique that uses specific antibodies to detect a single target protein (Maity, Sheff, & Fisher, 2013). The antibodies bound to the protein are then detected using a secondary antibody that allows for the visualization of the target (Maity, Sheff, & Fisher, 2013). Specifically, immunofluorescence staining uses fluorophore-conjugated antibodies to visualize the target protein (Maity, Sheff, & Fisher, 2013). In this paper by Zhang et al. (2020), the primary antibody against SARS-CoV-2 S protein was incubated with lung tissue of placebo and immunized mice. Detection of S protein was then visualized via detection of TSA-dendronfluorophores (Zhang et al., 2020).

RNA In-Situ Hybridization Assay

RNA in-situ hybridization uses a labeled RNA strand that is complementary to the RNA sequence that is being located (Lee, Xiong, & Xiong, 2013). The target RNA sequence is able to be located in a specific portion of the tissue or the entire tissue itself (Lee, Xiong, & Xiong, 2013). In this study conducted by Zhang et al. (2020), RNA in-situ hybridization was used to visualize viral RNA copies produced by SARS-CoV-2 infection in placebo and immunized mice.

Hematoxylin and Eosin (H&E) Stain

An hematoxylin and eosin (H&E) stain is a commonly used and routine method for viewing cellular details of tissue structure (Rolls & Sampias, 2019). Hematoxylin is a dye that is used to visualize nuclear details in cells by attaching to the anionic components of tissues (Rolls & Sampias, 2019). Eosin, on the other hand, is used as a counterstain that allows for the differentiation between the cytoplasm and nuclei of cells (Rolls & Sampias, 2019). In this study done by Zhang et al. (2020), an H&E stain was performed on lung tissues collected from placebo and immunized mice to visualize lung pathology following challenge of a mouse-adapted SARS-CoV-2 strain.

The purpose of the Figure 1 experiments was to design a vaccine that encapsulated the SARS-CoV-2 receptor binding domain (RBD)-encoding mRNA into a lipid nanoparticle (LNP) for in vivo delivery (Zhang et al., 2020).

Zhang et al. first synthesized an mRNA sequence that encoded for the RBD of SARS-CoV-2 (amino acids 319-541) as their target antigen along with a 5’ and 3’ untranslated region and a poly-A tail. Another mRNA sequence that encoded a firefly luciferase (FLuc) signal peptide was also synthesized and used in later experiments, as shown in Figure 1A.

Figure 1A: Schematic of mRNA construct encoding for SARS-CoV-2 RBD (Zhang et al., 2020).

To ensure that the mRNA could be translated by cells, the RBD-encoding mRNA was transfected into HeLa, Huh7, HEK293T, and Vero cell lines and recombinant RBD expression was measured using a Western blot. A high expression of recombinant RBD was observed in all supernatants with up to 917.4 ng/mL of RBD in the HEK293F cell line, as shown in Figure 1B (Zhang et al., 2020).

Figure 1B: Western blot showing RBD expression in HeLa, Huh7, HEK293T, and Vero cell lines. Cells were transfected with RBD-encoding mRNA (2 μg/mL) and immunoblotted for 48 h post transfection (left). RBD expression in HEK293F cell line measured by ELISA (right) (Zhang et al., 2020).

Figure 1D: Competitive inhibiton assay between rRBD and VSV-based pseudovirus expressing SARS-CoV-2 S protein. Data are shown as mean ± SEM; unpaired t test.****p < 0.0001. (Zhang et al., 2020).

Figure 1C: Real-time RBD-ACE2 binding assay (Zhang et al., 2020).

After purifying the rRBD from supernatants, its binding affinity to recombinant human ACE2 was measured using a binding assay. Based on kinetics analysis using ForteBio Octet, rRBD has a high affinity to ACE2, as shown in Figure 1C. rRBD also best inhibited vesicular stomatitis virus (VSV)-based pseudovirus expressing the SARS-CoV-2 S protein from entering Huh7 cells compared to negative controls, bovine serum albumin (BSA), and RBD, as shown in Figure 1D (Zhang et al., 2020).

Immunostaining using FITC in HeLa cells after transfection of RBD-encoding mRNA and RBD expression showed that the RBD protein could be recognized by a panel of monoclonal antibodies. Also, the RBD protein could be detected when with sera from three recovering COVID-19 patients, as shown in Figure 1E (Zhang et al., 2020).

Figure 1E: Immunofluorescence staining of RBD (FITC, green) in HeLa cells 24 h after transfection with 2 μg/mL of RBD mRNA. RBD expression was recognized by a panel of SARS-CoV-2 specific monoclonal antibodies (left). Immunofluorescence staining of RBD (FITC, green) with convalescent sera from three COVID-19 patients (right). Nuclei was stained using DAPI (blue). Scale bar: 50 μm (Zhang et al., 2020).

Figure 1G: Cryo-TEM graph of ARCoV. Scale bar: 200nm (Zhang et al., 2020).

Figure 1F: Size measurements of ARCoV using dynamic light-scattering method (Zhang et al., 2020).

ARCoV was formulated by mixing RBD mRNA in aqueous solution with lipids in ethanol which lead to spontaneous formation of LNPs with mRNA encapsulated inside. Tangential flow filtration was used to concentrate and purify the solution before it was put into sterilized glass bottles or syringes. The final stock of ARCoV had an average particle size of 88.85 nm, as shown in Figure 1F. Cryo-transmission electron microscopy (TEM) showed that the LNPs are homogenous solid spheres that lack an aqueous interior, as shown in Figure 1G (Zhang et al., 2020).

The results of Figure 1 demonstrated that the RBD-encoding mRNA can express the RBD protein in a variety of cell types and the RBD protein has a high resemblance to wild-type RBD. The RBD protein can also be detected in sera and by monoclonal antibodies and ARCoV has a homogenous appearance after formulation (Zhang et al., 2020).

The purpose of Figure 2 experiments was to discover the absorption and distribution of ARCoV in mice model.

To visualize the distribution of the lipid nanoparticles in mice bodies, a firefly luciferase encoding mRNA was packed in the nanoparticles, and underwent intramuscular and subcutaneous injection into the mice. It was then visualized using bioluminescence analysis. In Figure 2A, intramuscular and subcutaneous injection both resulted in robust expression of the luciferase in the upper abdomen of the mice body. The third delivery method, intranasal inoculation did not lead to any expression of the protein. In Figure 2B, the organs were removed to complete an ex vivo analysis, and results showed that the most robust expression was seen in the liver. The muscle and spleen also gave slight signals of fluorescence, which indicated that the content of the nanoparticles would be expressed most predominantly in the liver. Figure 2C shows a comparison between receptor binding domain concentrations of placebo and ARCoV immunized mice. It showed that the RBD concentration of immunized mice was significantly higher than that of the placebo (Zhang et al., 2020).

Figure 2ABC: Figure 2A displays the in vivo BLI of reporter mRNA-LNP in female BALB/c mice, which were inoculated intramuscularly, subcutaneously, and intranasally. Figure 2B shows the tissue distribution of mRNA-LNP in vaccinated mice, with empty LNP as a control. Figure 2C shows the expression of mRNA-encoded RBD in mice. Mice serum was measured by ELISA 6h post-inoculation. (Zhang et al., 2020).

Figure 2D and Figure 2E are multiplex immunostaining analyses of the mice muscle and liver tissue, respectively. This analysis was performed to identify the cell types that express the RBD. The ARCoV expressing site in muscle sample was predominantly colonized by CD11b-positive monocytes, CD163-positive macrophages, and CD103-positive dendritic cells, as seen in Figure D. Figure E indicates that glutamine synthetase-positive pericentral hepatocytes surrounding the CD31- positive central vein (CV), Arg1-positive hepatocytes, and CD163-positive liver macrophages are recruited at the site in the liver that expresses the mRNA (Zhang et al., 2020).

Figure 2E: Mouse liver expression of mRNA-LNP. Liver tissue was collected 6h post-vaccination. Tissue was stained for multiple markers including SARS-CoV-2 RBD, glutamine synthetase, CD31, CD163, and Arg1 (Zhang et al., 2020).

Figure 2D: Multiplex immunostaining analysis for expression of LNP-delivered mRNA in muscle tissue of female BALB/c immunized mice. Muscle tissue was collect 6h post inoculation, and stained for various cell markers (Zhang et al., 2020).

The results from experiments conducted in Figure 2 suggest that intramuscular and subcutaneous injection of ARCoV results in mRNA expression in the upper abdomen, and efficiently recruits immune cells (Zhang et al., 2020).

The purpose of the Figure 3 experiments was to determine the humoral immune response in ARCoV-vaccinated mice (Zhang et al., 2020).

Figure 3A highlights the immunization, sample collection, and challenge schedule for the following experiments. Female BALB/c mice received their first immunization on day 0, and a second immunization on day 14. Samples of serum were collected each week following the initial vaccination on days 7, 14, 21 and 28, to be used for antibody detection experiments. Serum collected on day 28 was also used for T-cell detection. The mice were challenged with SARS-CoV-2 on day 40. Tissue was then harvested on day 45 (Zhang et al., 2020).

Figure 3A: Schematic for the immunization, sample collection, and challenge schedule of BALB/c mice receiving ARCoV (Zhang et al., 2020).

Figure 3C: NT50 neutralizing antibody titer of ARCoV-vaccinated mice determined by VSV-based pseudovirus neutralization assay (Zhang et al., 2020).

Figure 3B: Results of an ELISA used to determine the SARS-CoV-2-specific IgG antibody titer in ARCoV-vaccinated mice (Zhang et al., 2020).

Figure 3B represents the results of an enzyme-linked immunosorbent assay (ELISA) used to detect the presence of antibodies in the serum of vaccinated mice. The specific antibody being detected in this assay was SARS-CoV-2 RBD-specific IgG. Elevated IgG is important in the development of long-term immunity following vaccination. This graph compares control mice given placebo (an empty lipid nanoparticle) with mice that were vaccinated with 2 μg and 10 μg of ARCoV. As expected, the placebo did not elicit production of detectable levels of virus-specific IgG, but vaccinated mice that received either dose (2 μg or 10 μg) had elevated levels of IgG in their serum, which was most evident after day 14 (Zhang et al., 2020).

The following two experiments measured the neutralizing antibody titers in BALB/c mice vaccinated with ARCoV. Neutralizing antibodies are of particular interest because of their ability to block virus entry into host cells. Figure 3C displays the results of a pseudovirus-based neutralization assay, used to determine the neutralizing antibody titer (NT50) resulting from ARCoV vaccination. The NT50 was defined as the dilution at which the relative light units (RLU) were reduced by 50% compared to the control virus. On day 28, the NT50 titers approached 1/2540 in the mice that received the 2 μg vaccine, and 1/7079 in the mice that received the 10 μg vaccine (Zhang et al., 2020).

Figure 3D shows the neutralizing antibody titers resulting from a plaque reduction neutralization test (PRNT50). The PRNT50 was defined as the dilution that resulted in a reduction of plaque forming units (PFU) by 50%. Plaque forming units appear as an area of clearing on a plate where the number of PFU is inversely proportional to the level of neutralizing antibodies present in the sera of a vaccinated mouse. Results of this assay showed that the PRNT50 reached 1/2194 in the serum of mice vaccinated with 2 μg and 1/5704 in the serum of mice vaccinated with 10 μg of ARCoV (Zhang et al., 2020).

Figure 3D: Neutralizing antibody titer of ARCoV-vaccinated mice determined by plaque-reduction neutralization test (Zhang et al., 2020).

Figure 3E: Cross-neutralization of ARCoV-vaccinated mice serum across three strains of SARS-CoV-2: 131, V34, and 5N. Results were analyzed by one-way ANOVA (Zhang et al., 2020).

Due to the prevalence of multiple strains of SARS-CoV-2, Zhang et al. wanted to determine the neutralizing effect of serum from vaccinated mice against three different strains of the virus. Several strains of mutated SARS-CoV-2 have been reported, some of which have mutations in the spike protein that the mRNA for ARCoV was obtained from, and this could affect transmission and pathogenesis of the virus (Korber et al., 2020). Figure 3E displays the comparison of the PRNT50 obtained from testing the antibody obtained from serum of vaccinated mice, against three strains of the virus, 131, V34, and 5N. Results showed that the neutralizing ability of serum from vaccinated mice was similar against all three strains tested, as a significant difference was not detected in the PRNT50 titers (Zhang et al., 2020).

The overall conclusion from the experiments described in Figure 3 was that two doses of the ARCoV vaccine in mice was capable of inducing high levels of antibodies with neutralizing ability against SARS-CoV-2 (Zhang et al., 2020).

The purpose of the experiments shown in Figure 4 was to determine whether intramuscular vaccination with two doses of ARCoV would result in a SARS-CoV-2-specific T-cell response in mice (Zhang et al., 2020).

Flow cytometry was conducted to detect the presence of CD4⁺ and CD8⁺ effector memory T (T_EM) cells, which are important for long-term protection against the virus. The results in Figure 4A show that there were significantly elevated levels of CD4⁺ and CD8⁺ T_EM cells in vaccinated mouse splenocytes compared to the mice vaccinated with the placebo lipid nanoparticles (Zhang et al., 2020). Both types of T cells have specific functions in the immune system:

• CD8⁺ cytotoxic T cells: recognize peptides from MHC Class I. They important for immune defense against intracellular pathogens including viruses and bacteria, and they secrete TNF-alpha, and IFN-gamma (Wissinger, n.d.).
• CD4⁺ helper T cells: recognize peptides from MHC Class II. There are two types – Th1 and Th2. Th1 cells are responsible for controlling intracellular pathogens (i.e. viruses and some bacteria). Th2 cells are involved in defense against large extracellular organisms and are involved in allergies (Bell, n.d.).

Figure 4A: SARS-CoV-2 RBD-specific CD4+ and CD8+ T effector memory cells detected by flow cytometry in ARCoV-vaccinated mouse splenocytes (Zhang et al., 2020).

Next, Zhang et al. indirectly measured the number cytokine-secreting cells in the splenocytes of ARCoV-vaccinated mice, by detecting cytokines in an ELISPOT assay. The first set of cytokines tested were IFN-γ, TNF-α, and IL-2, which are secreted by Th1 cells. Figure 4B shows that after quantifying the spot forming cells (SFC) per million cells, there was a significantly higher secretion of IFN-γ, TNF-α, and IL-2 in the splenocytes of vaccinated mice compared to the placebo mice (Zhang et al., 2020).

Figure 4B: Results of ELISPOT assay performed on serum of ARCoV-vaccinated mice for detection of IFN-γ, TNF-α, and IL-2 (Zhang et al., 2020).

The next set of cytokines tested in the ELISPOT assay were IL-4 and IL-6, which are secreted by Th2 cells and macrophages respectively. Figure 4C shows that there was no significant difference in IL-4 and IL-6 levels between ARCoV-vaccinated and placebo mice after quantifying SFC per million cells (Zhang et al., 2020).

Figure 4C: Results of ELISPOT assay performed on serum of ARCoV-vaccinated mice for detection of IL-4 and IL-6 (Zhang et al., 2020).

The overall results of the experiments described in Figure 4 demonstrate that the ARCoV vaccine induces a SARS-CoV-2-specific Th1-biased immune response (Zhang et al., 2020).

The purpose of the experiments shown in Figure 5 was to further evaluate the protection that ARCoV provides in vivo following SARS-CoV-2 challenge in mice (Zhang et al., 2020).

In this set of experiments, a new SARS-CoV-2 mouse-adapted strain was developed and used to challenge immunocompetent BALB/c mice (Zhang et al., 2020). Mice received two doses of ARCoV mRNA-LNP at either 2μg or 10μg and were subsequently challenged with MASCp6, a mouse-adapted SARS-CoV-2 strain (Zhang et al., 2020). Mice were then euthanized 5 days following challenge, and sections of their lung and trachea were analyzed (Zhang et al., 2020). Upon intranasal challenge of MASCp6, robust viral replication in the lungs and trachea of placebo mice was observed, indicated by the high level of viral RNA copies shown in Figure 5A and 5B (Zhang et al., 2020). However, following intranasal challenge of MASCp6 in mice immunized with either 2μg or 10μg of ARCoV, no measurable viral RNA was detected in the lungs or trachea (Zhang et al., 2020). This shows that all mice immunized with 2μg or 10μg of ARCoV showed full protection against SARS-CoV-2 challenge, which is consistent with the high neutralizing antibody titers produced and measured in the previous experiments (Zhang et al., 2020).

Figure 5AB: Viral RNA loads in lung and trachea of placebo and ARCoV immunized mice following MASCp6 challenge (Zhang et al., 2020).

Next, the lung sections of placebo and immunized mice were analyzed and compared using histopathological assays. First, an immunostaining assay was performed with a SARS-CoV-2 S-specific monoclonal antibody (Zhang et al., 2020). Placebo lung sections showed significant SARS-CoV-2 protein expression, shown in Figure 5C, compared to sections from mice immunized with 2 or 10 ug of ARCoV (Zhang et al., 2020). An in-situ hybridization assay was also performed, where labeled complementary RNA is used to localize specific RNA sequences (Zhang et al., 2020). This assay detected SARS-CoV-2 specific RNA in placebo mice but no viral RNA in ARCoV immunized mice, as shown in Figure 5D (Zhang et al., 2020). Lastly, a H&E staining was used to visualize lung pathology in Figure 5E (Zhang et al., 2020). The placebo group, which were vaccinated with empty LNPs, developed typical lung lesions associated with infection of SARS-CoV-2 (Zhang et al., 2020). This is characterized by denatured epithelial tissues, thickened alveolar septa, and activated inflammatory cell infiltration (Zhang et al., 2020). This contrasted with the lung samples of immunized mice, which showed no pathological changes (Zhang et al., 2020).

Figure 5CDE: Immunostaining, in-situ hybridization, and H&E staining of lung sections in placebo and ARCoV immunized mice (Zhang et al., 2020).

The overall results of the experiments described in Figure 5 demonstrate that two doses of ARCoV vaccination completely prevent SARS-CoV-2 replication in the lower respiratory tract and protect mice from lung lesions (Zhang et al., 2020).

The purpose of the experiments shown in Figure 6 was to determine and compare serum neutralizing antibody titers of ARCoV-vaccinated mice before and after SARS-CoV-2 challenge (Zhang et al., 2020).

In this next experiment, either 2 doses of 2μg or 10μg of ARCoV was administered to mice, as shown in Figure 6A (Zhang et al., 2020). However, there was no significant increase in neutralizing antibody titers following challenge with SARS-CoV-2, as indicated by the NT50 values (Zhang et al., 2020).

Figure 6A: NT50 values before and after SARS-CoV-2 challenge in mice vaccinated with 2 doses of 2μg or 10μg of ARCoV (Zhang et al., 2020).

However, as shown in Figure 6B, mice that received a single ARCoV dose of 2μg or 30μg sustained an increase in NT50 after challenge (Zhang et al., 2020). This increase in neutralizing antibodies indicates an induction of a memory immune response, which results in a renewed and rapid production of antibodies against the pathogen, following secondary exposure (Zhang et al., 2020).

Figure 6B: NT50 values before and after SARS-CoV-2 challenge in mice vaccinated with a single dose of 2μg or 30μg of ARCoV (Zhang et al., 2020).

Figures 6C and 6D show the correlation between viral RNA copies found in the lungs and protective efficacy of ARCoV via NT50 and PRNT50 values in ARCoV-vaccinated mice (Zhang et al., 2020). These results show that PRNT50 and NT50 titers are inversely correlated with lung viral RNA loads, where higher PRNT50 and NT50 values are associated with lower viral RNA copies (Zhang et al., 2020). This suggests that higher levels of neutralizing antibodies induced by ARCoV vaccination provides protection against SARS-CoV-2 challenge, as indicated by lower viral RNA copies.

Figure 6CD: Correlations of viral loads and protective efficacy of ARCoV via PRNT50 and NT50 values (Zhang et al., 2020).

This set of experiments therefore suggests that vaccine-elicited serum neutralizing antibody titers can be representative of protection against SARS-CoV-2 challenge (Zhang et al., 2020).

The purpose of the experiments shown in Figure 7 was to determine the immunogenicity of ARCoV in cynomolgus macaques (Zhang et al., 2020).

Figure 7A: ARCoV immunization, serum and antibody collection and T cell immunity schedule of cynomolgus monkeys receiving ARCoV (Zhang et al., 2020).

In this experiment, researchers examined the efficacy of ARCoV in cynomolgus monkeys' bodies. Figure 7B and 7C illustrate the IgG titer and neutralization titer 50 of the monkeys 14 and 28 days after vaccinations of 100ug and 1000ug doses, and significant increase in both values were observed, meaning that its efficiency of inducing immune response was confirmed. (Zhang et al., 2020)

Figure 7B and 7C: IgG titer and neutralization titer of cynomolgus monkeys 0, 14 and 28 days after vaccinated with placebo, 100ug and 1000ug of ARCoV respectively. n.s. indicates not-significant differences and star signs indicate significant changes. (Zhang et al., 2020).

Further experiments, as shown in Figure 7D and 7E were performed to examine whether the immune response triggered by the vaccine is Th1 or Th2 biased response. The experiment was designed to test the level of gamma-interferon, which is produced by CD4 Th1 cells, and IL-4 which induces differentiation of naïve helper T cell to Th2 cells. The result shows that the gamma-interferon level was increased significantly after vaccination while the IL-4 level remained unchanged, indicating Th1 biased immunity was triggered. (Zhang et al., 2020)

Figure 7D and 7E: Comparison of the increases in gamma-interferon and IL-4 levels in monkeys’ bodies after different doses of vaccination. (Zhang et al., 2020).

This set of experiments suggest that the vaccine efficiently induce Th1 biased immunity in cynomolgus monkeys’ bodies.(Zhang et al., 2020)

This study demonstrated the immunogenicity and efficacy of an mRNA-LNP vaccine against mouse-adapted SARS-CoV-2 in animal models (Zhang et al., 2020). A single dose or two doses was able to elicit a humoral and cellular immune response in mice and non-human primates when challenged with SARS-CoV-2 (Zhang et al., 2020). In addition, the mRNA-LNP vaccine that was constructed was well formulated which is evident from the paper’s dynamic light scattering data and cryo-TEM graph of their LNPs. When this vaccine was introduced intramuscularly and subcutaneously into mouse models, mRNA expression could be detected in the upper abdomen and several immune cells were efficiently recruited. The vaccine also elicited a Th1-biased SARS-CoV-2-specific cellular immune response which is important in preventing vaccine-associated enhanced respiratory disease (VAERD) (Zhang et al., 2020). VAERD is a phenomenon where a patient has an unusual presentation of an infection from a pathogen that they were previously vaccinated for (World Health Organization, 2020). Not only did the vaccine provided protection in mouse subjects, but it also completely prevented SARS-CoV-2 replication in the lower respiratory tract and protected mice from lung lesions (Zhang et al., 2020). These robust results show that ARCoV may be a viable COVID-19 vaccine candidate.

While this paper did have many new findings, it did have some limitations. In this study, Zhang et al. (2020) used a mouse adapted strain of SARS-CoV-2 instead of wild-type SARS-CoV-2 so it’s unknown how effective this vaccine would be in humans. Moving forward, they can do further experiments using wild-type SARS-Cov-2 in transgenic ACE2 mice and non-human primates to test the vaccine’s efficacy (Zhang et al., 2020). Another limitation is that the duration of neutralizing antibodies is unknown. Previous research has shown reinfection of coronaviruses after vaccination (Callow et al., 1990; Wu et al., 2007) so there is a risk of reinfection of SARS-CoV-2 after vaccination if immunity is short-term (Zhang et al., 2020). Therefore, it is important to evaluate long-term immunity in animal models and eventually humans (Zhang et al., 2020). Now that ARCoV is in phase 1 clinical trials (Zhang et al., 2020), this vaccine must be tested for safety in humans before moving on to further clinical trials that test for efficacy. Finally, long-term stability assays must be performed to ensure that ARCoV is stable under a variety of conditions (Zhang et al., 2020).

Despite the limitations, this paper (Zhang et al., 2020) has provided a number of contributions in studying SARS-CoV-2. So far, this paper has been cited over thirty times by other researchers. Many of these are review papers that discuss mRNA vaccines (Wang Y. et al., 2020) or the use of LNPs (Aldosari et al., 2021) or primary research articles that are in pre-clinical trials with their own vaccine (Elia et al., 2021; Wang W. et al., 2021; Huang et al., 2021). One interesting example is a study by Elia et al. (2021) where they designed their own mRNA-LNP vaccine but they focused on how the composition of the LNP would affect delivery and immunogenicity.

There are also a number of things that this paper discovered that weren’t known previously. First, this was the first study in clinical trials to show immunogenicity and efficacy of an mRNA vaccine on animal models (Zhang et al., 2020). In previous research it has been discussed that VAERD can occur due to Th2-biased CD4+ T cell responses. This is a phenomenon where a person shows an unusual presentation of an infection after being vaccinated for the pathogen that causes that infection. Luckily, this vaccine induced a Th1-biased T cell response so no enhanced risk of viral replication or clinical disease was present even after 1 dose of vaccine (Zhang et al., 2020). Moreover, compared to the full length S protein in the Moderna vaccine, this RBD vaccine has a lower risk of inducing non-neutralizing antibodies which lowers the risk of antibody-dependent enhancement (Zhang et al., 2020). This is when antibodies actually aid in viral entry. Finally and most importantly, due to its thermostability, this vaccine can be stored at 4°C and 25°C for at least 7 days (Zhang et al., 2020), giving it a major advantage over the Pfizer and Moderna mRNA vaccines. In conclusion, this paper has major implications in the understanding and development of mRNA vaccines against SARS-CoV-2.

Aldosari, B. N., Alfagih, I. M., & Almurshedi, A. S. (2021). Lipid Nanoparticles as Delivery Systems for RNA-Based Vaccines. Pharmaceutics, 13(2), 206. https://doi.org/10.3390/pharmaceutics13020206

Bell, L. (n.d.). CD4+ T Cells | British Society for Immunology. Retrieved February 27, 2021, from https://www.immunology.org/public-information/bitesized-immunology/cells/cd4-t-cells

Callow, K. A., Parry, H. F., Sergeant, M., & Tyrrell, D. A. (1990). The time course of the immune response to experimental coronavirus infection of man. Epidemiology and infection, 105(2), 435–446. https://doi.org/10.1017/s0950268800048019

Chu, H., Chan, J. F-W., Yuen T. T-T., Shuai, H., Yuan, S., Wang, Y., . . . Yuen, P. K-Y. (2020). Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study. The Lancet, 1(1), E14-E23. DOI:https://doi.org/10.1016/S2666-5247(20)30004-5

Creative Biolabs. (n.d.). Biolayer Interferometry (BLI). Creative Biolabs. https://www.creative-biolabs.com/drug-discovery/therapeutics/biolayer-interferometry-bli.htm

Elia, U., Ramishetti, S., Rosenfeld, R., Dammes, N., Bar-Haim, E., Naidu, G. S., Makdasi, E., Yahalom-Ronen, Y., Tamir, H., Paran, N., Cohen, O., & Peer, D. (2021). Design of SARS-CoV-2 hFc-Conjugated Receptor-Binding Domain mRNA Vaccine Delivered via Lipid Nanoparticles. ACS nano, acsnano.0c10180. Advance online publication. https://doi.org/10.1021/acsnano.0c10180

ELISpot Assay Principle. (n.d.). Retrieved February 28, 2021, from https://www.mabtech.com/knowledge-center/assay-principles/elispot-assay-principle

Government of Canada. (2021). Vaccines for COVID-19: Authorized vaccines. Canada.ca. https://www.canada.ca/en/health-canada/services/drugs-health-products/covid19-industry/drugs-vaccines-treatments/vaccines.html

Gu, H., Chen, Q., Yang, G., He, L., Fan, H., Deng, Y-Q., . . . Zhou, Y. (2020). Rapid adaptation of SARS-CoV-2 in BALB/c mice: Novel mouse model for vaccine efficacy. bioRxiv. https://doi.org/10.1101/2020.05.02.073411

Horlock, C. (n.d.). Enzyme-linked immunosorbent assay (ELISA). British Society of Immunology. https://www.immunology.org/public-information/bitesized-immunology/experimental-techniques/enzyme-linked-immunosorbent-assay

How a Flow Cytometer Works | Thermo Fisher Scientific - CA. (n.d.). Retrieved February 28, 2021, from https://www.thermofisher.com/ca/en/home/life-science/cell-analysis/cell-analysis-learning-center/molecular-probes-school-of-fluorescence/flow-cytometry-basics/flow-cytometry-fundamentals/how-flow-cytometer-works.html

Huang, Q., Ji, K., Tian, S., Wang, F., Huang, B., Tong, Z., Tan, S., Hao, J., Wang, Q., Tan, W., Gao, G. F., & Yan, J. (2021). A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2. Nature communications, 12(1), 776. https://doi.org/10.1038/s41467-021-21037-2

Jackson, L. A., Anderson, E. J., Rouphael, N. G., Roberts, P. C., Makhene, M., Coler, R. N., McCullough, M. P., Chappell, J. D., Denison, M. R., Stevens, L. J., Pruijssers, A. J., McDermott, A., Flach, B., Doria-Rose, N. A., Corbett, K. S., Morabito, K. M., O'Dell, S., Schmidt, S. D., Swanson, P. A., . . . Beigel, J. H. (2020). An mRNA Vaccine against SARS-CoV-2 — Preliminary Report. New England Journal of Medicine, 383(20), 1920–1931. https://doi.org/10.1056/NEJMoa2022483

Jackson, N. (2020, September 4). Vessels for Collective Progress: the use of HeLa cells in COVID-19 research. SITN. https://sitn.hms.harvard.edu/flash/2020/vessels-for-collective-progress-the-use-of-hela-cells-in-covid-19-research/#:~:text=HeLa%20cells%20do%20not%20naturally,begin%20their%20cycle%20of%20infection.

Kaye M. (2006). SARS-associated coronavirus replication in cell lines. Emerging infectious diseases, 12(1), 128–133. https://doi.org/10.3201/eid1201.050496

Korber, B., Fischer, W. M., Gnanakaran, S., Yoon, H., Theiler, J., Abfalterer, W., … Montefiori, D. C. (2020, April 30). Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2. BioRxiv. bioRxiv. https://doi.org/10.1101/2020.04.29.069054

Lee, D., Xiong, S., &amp; Xiong, W. (2013). General introduction to in situ hybridization protocol using nonradioactively labeled probes to detect mRNAs on tissue sections. Methods in Molecular Biology, 1018, 165-174. doi:10.1007/978-1-62703-444-9_16.

Li, X. F., Dong, H. L., Wang, H. J., Huang, X. Y., Qiu, Y. F., Ji, X., … Qin, C. F. (2018). Development of a chimeric Zika vaccine using a licensed live-attenuated flavivirus vaccine as backbone. Nature Communications, 9(1), 1–11. https://doi.org/10.1038/s41467-018-02975-w

Maity, B., Sheff, D., &amp; Fisher, R. A. (2013). Immunostaining: Detection of signaling protein location in tissues, cells and subcellular compartments. Methods in Cell Biology, 113, 81-105. doi:10.1016/B978-0-12-407239-8.00005-7

Matsuyama, S., Nao, N., Shirato, K., Kawase, M., Saito, S., Takayama, I., . . . Takeda, M. (2020). Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells. PNAS, 117(13), 7001-7003. https://doi.org/10.1073/pnas.2002589117

MGI - Inbred Strains: BALB. (n.d.). Retrieved February 27, 2021, from http://www.informatics.jax.org/inbred_strains/mouse/docs/BALB.shtml Nie, J., Li, Q., Wu, J., Zhao, C., Hao, H., Liu, H., … Wang, Y. (2020). Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2. Emerging Microbes and Infections, 9(1), 680–686. https://doi.org/10.1080/22221751.2020.1743767

Mirabelli, C., Wotring, J. W., Zhang, C. J., McCarty, S. M., Fursmidt, R., Frum, T., . . . Sexton, J. Z. (2020). Morphological Cell Profiling of SARS-CoV-2 Infection Identifies Drug Repurposing Candidates for COVID-19. bioRxiv, Preprint, NaN NaN. doi: 10.1101/2020.05.27.117184

Mulligan, M. J., Lyke, K. E., Kitchin, N., Absalon, J., Gurtman, A., Lockhart, S., Neuzil, K., Raabe, V., Bailey, R., Swanson, K. A., Li, P., Koury, K., Kalina, W., Cooper, D., Fontes-Garfias, C., Shi, P., Tureci, O., Tompkins, K. R., Walsh, E. E., . . . Jansen, K. U. (2020). Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adults. Nature, 586(7830), 589–593. https://doi.org/10.1038/s41586-020-2639-4

Nie, J., Li, Q., Wu, J., Zhao, C., Hao, H., Liu, H., Zhang, L., Nie, L., Qin, H., Wang, M., Lu, Q., Li, X., Sun, Q., Liu, J., Fan, C., Huang, W., Xu, M., & Wang, Y. (2020). Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2. Emerging microbes & infections, 9(1), 680–686. https://doi.org/10.1080/22221751.2020.1743767

Pardi, N., Hogan, M. J., Porter, F. W., & Weissman, D. (2018). mRNA vaccines-a new era in vaccinology. Nat. Rev. Drug Discov., 17,(4) 261-279. https://doi.org/10.1038/nrd.2017.243

Plaque Reduction Neutralization Tests (PRNT). (n.d.). Retrieved February 28, 2021, from https://www.labce.com/spg1050613_plaque_reduction_neutralization_tests_prnt.aspx

Pseudovirus Neutralization Assays in SARS-CoV-2 Research - Berthold Technologies. (n.d.). Retrieved February 28, 2021, from https://www.berthold.com/en/bioanalytic/solutions-sars-cov-2-covid-19-research/pseudovirus-neutralization-assays-in-sars-cov-2-research/

Rolls, G., &amp; Sampias, C. (2019, August 14). H&amp;e staining overview: A guide to best practices. Retrieved February 28, 2021, from https://www.leicabiosystems.com/knowledge-pathway/he-staining-overview-a-guide-to-best-practices/

Stetefeld, J., McKenna, S. A., & Patel, T. R. (2016). Dynamic light scattering: a practical guide and applications in biomedical sciences. Biophysical reviews, 8(4), 409–427. https://doi.org/10.1007/s12551-016-0218-6

Thermo Fisher Scientific. (n.d.). Overview of ELISA. Thermo Fisher Scientific. https://www.thermofisher.com/ca/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa.html

Wang, W., Huang, B., Zhu, Y., Tan, W., & Zhu, M. (2021). Ferritin nanoparticle-based SARS-CoV-2 RBD vaccine induces a persistent antibody response and long-term memory in mice. Cellular & molecular immunology, 1–3. Advance online publication. https://doi.org/10.1038/s41423-021-00643-6

Wang, Y., Zhang, Z., Luo, J., Han, X., Wei, Y., & Wei, X. (2021). mRNA vaccine: a potential therapeutic strategy. Mol Cancer, 20(33). doi: 10.1186/s12943-021-01311-z

What is an ELISA? (n.d.). R&D Systems. https://www.rndsystems.com/resources/what-is-an-elisa-and-elisa-types#:~:text=In%20a%20direct%20ELISA%2C%20an,of%20analyte%20in%20the%20sample.

World Health Organization. (2020, October 30). 21st WHO Regulatory Update on COVID-19. World Health Organization. https://www.who.int/publications/m/item/21st-who-regulatory-update-on-covid-19

Wissinger, E. (n.d.). CD8+ T Cells | British Society for Immunology. Retrieved February 27, 2021, from https://www.immunology.org/public-information/bitesized-immunology/cells/cd8-t-cells

Wu, L., Wang, N., Chang, Y., Tian, X., Na, D., Zhang, L….Liang, G. (2007). Duration of Antibody Responses after Severe Acute Respiratory Syndrome. Emerging Infectious Diseases, 13(10), 1562-1564. https://dx.doi.org/10.3201/eid1310.070576.

Yakimchuk, K. (2011, January 19). Receptor-Ligand Binding Assays. Labome.com. https://www.labome.com/method/Receptor-Ligand-Binding-Assays.html

Zhang, N. N., Li, X. F., Deng, Y. Q., Zhao, H., Huang, Y. J., Yang, G., … Qin, C. F. (2020). A Thermostable mRNA Vaccine against COVID-19. Cell, 182(5), 1271-1283.e16. https://doi.org/10.1016/j.cell.2020.07.024