FIELD OF INVENTION

[0001] Present invention relates to COVID-19 vaccines, and particularly to peptide-based vaccines comprising highly conserved epitopes from the spike protein of SARS-CoV-2.

BACKGROUND

[0002] SARS-CoV-2 became a significant global health problem, causing COVID-19 pandemic in March 2020. Because vaccines are the most economical means to control infectious diseases, broad and effective vaccines against SARS-CoV-2 infections are urgently needed.

[0003] Like MERS-CoV and SARS-CoV, SARS-CoV-2 is a positive-strand RNA virus that belongs to the group of beta-coronaviruses. The genome of SARS-CoV-2 encodes 4 major structural proteins, including a spike (S) protein, an envelope (E) protein, a membrane (M) protein, and a nucleocapsid (N) protein. SARS-CoV-2 enters host cells, primarily lung epithelial cells, through a catch-and-fuse process that involves interactions between its S protein and human angiotensin-converting enzyme 2 (ACE2) expressed on host cells.

[0004] FIG. 1 shows a schematic illustrating a possible mechanism for SARS-CoV-2 entry into the host cells. First, the spike (S) protein binds the ACE2 receptor on the host cells. Then, a protease transforms the spike protein. As a result, the spike protein penetrates the host cells and brings the membrane of the host cell and the viral membrane closer. When the membranes fuse, it creates a pore for the viral genome to enter the host cells.

[0005] In the virus entry processes, the spike protein S is cleaved into an N-terminal S 1 region and a C-terminal S2 region. These two sub-units mediate distinct tasks of attachment and entry into the host cells. Specifically, SI mediates the binding to ACE2 to allow the virus to attach to the host cells. On the other hand, S2 mediates fusion of viral and host cell membranes to create a pore for viral genome entry into the host cells. The S2 domain is further cleaved at the S2’ site to separate a fusion peptide (FP) from the internal fusion peptide, and the fusion peptide then participates in the viral entry process.

[0006] Being important for viral attachment and entry into the host cells, the S protein or antibodies against the receptor-binding domain (RBD) contained in the S protein can serve as an immunogen to induce protective immunity. That is, antibodies against the S protein or antibodies against the receptor-binding domain (RBD) contained in the S protein may be able to prevent viral infections of the host cells. However, it has been reported that the SARS-CoV-2 S 1 domain, which contains the RBD, is frequently mutated, which can potentially make vaccines less effective or completely ineffective. Thus, an important consideration in the vaccine design is to use a conserved antigen that can generate universal antibodies to prevent infection by SAR.S- CoV-2 variants.

SUMMARY

[0007] Embodiments of the invention relate to the use of S2’ -peptides of SAR.S-CoV-2 as vaccine antigens or immune boosters. While the spike protein (S protein) of SAR.S-CoV-2 or the receptor-binding domain (RBD) in the spike protein of SAR.S-CoV-2 are popular targets for vaccine developments, inventors of the present invention have found that the S2’ -peptides, which surround the S2’ protease cleavage site, of SAR.S-CoV-2 provide more attractive targets. The S2’ peptides are highly conserved among different coronavirus species, suggesting that these S2’ peptide sequences may play critical roles (e.g., membrane fusion and cell entry) for coronaviruses. Therefore, the S2’ peptides are less likely to have mutations in the ever-evolving variants of SAR.S-CoV-2. Inventors of the invention have found that blocking the fusion domain of coronavirus using vaccines targeting the enzymatic S2’ -cleavage site can disrupt the virus entry processes, thereby neutralizing viral entry into the host cells. In addition, inventors of the present invention have unexpected found that these S2’-peptides also can be used as agonist adjuvants, immune boosters, or carrier epitopes in vaccine developments.

[0008] One aspect of the invention relates to vaccines for generation of immunity against SAR.S-CoV-2 infection. In accordance with one embodiment of the invention, a vaccine for generation of immunity against SAR.S-CoV-2 infection comprises an S2’ -peptide of SAR.S- CoV-2 in a formulation that enhances immune responses, wherein the S2’ -peptide comprises the amino acid sequence selected from SEQ ID NO: 1-10.

[0009] In accordance with embodiments of the invention, the formulation that enhances immune responses may comprise nanocomplexes encapsulating the S2’ -peptide, or the formulation includes TREM-like transcript- 1 (TREMLl) extracellular domain (ECD) or a stalk peptide as an immune booster. In accordance with embodiments of the invention, the nanocomplexes may comprise poly- g-glutamic acid (g-PGA) and chitosan. The TREM-like transcript-1 (TREMLl) extracellular domain (ECD) or stalk peptide may comprise the amino acid sequence selected from SEQ ID NO: 11-14. [0010] One aspect of the invention relates to pharmaceutical compositions for use in boosting an immune response. In accordance with one embodiment of the invention, a pharmaceutical composition for use in boosting an immune response comprises an S2’ -peptide of SARS-CoV- 2, wherein the S2’-peptide comprises the amino acid sequence selected from SEQ ID NO: 1-10.

[0011] Other aspects of the invention would become apparent from the following detailed description and the accompanying drawings.

BRIEF DESCROPTION OF THE DRAWINGS

[0012] FIG. 1 shows a schematic illustration of a catch-and-fuse process of coronavirus entry into host cells. First, the spike (S) protein of the coronavirus binds the ACE2 receptor on the host cells. A protease then transforms the spike protein, allowing the spike protein to penetrate the host cells and bring the membrane of the host cell and the viral membrane closer. After membrane fusion, a pore is formed to allow the viral genome to enter the host cells.

[0013] FIG. 2 shows a sequence alignment of S2’ cleavage sites between SARS-CoV, and SARS-CoV-2. As shown in FIG. 2, the fusion peptides and the protease cleavage sites are conserved between these viruses. These conserved sequences confer important functions in the host cell entry processes. While other parts of the viral genome are prone to mutations, these conserved fusion peptide sequences are unlikely to harbor any mutation, making them ideal as immunogens for vaccine development.

[0014] FIG. 3 shows a schedule of vaccinations, and blood samplings using a BALB/c mice model fortesting the immunogenicity of S2’ peptides. BALB/c mice were immunized with either nanocomplexes (NCs) only, S2’-l/NCs, triggering receptor expressed on myeloid cell-like transcript 1 (TREMLl) extracellular domain (ECD) only, spike protein receptor binding domain (RBD)/TREML 1 ECD, S2’-6/TREMLl ECD, or RBD/TREMLl ECD+ S2’-6/TREMLl ECD (n=5-6/group). Mouse sera were collected at day 42.

[0015] FIG. 4 shows serum antibody titers for the S2’ peptide-specific IgGl (serum dilution of 1:10000) and IgG2a (serum dilution of 1:1000) serum levels upon inoculations with S2’ peptide vaccines. Data are shown as S/N ratio.

[0016] FIG. 5 shows that the S2’-6/TREMLl ECD and S2’-l/NCs vaccines induce high titers of anti-spike (ECD) IgG antibodies in BALB/c mice. The results were presented as endpoint dilution serum titer. [0017] FIG. 6 shows RBD-specific IgGl and IgG2a serum levels upon inoculations with RBD/TREMLl ECD vaccine or RBD/TREMLl ECD plus S2’-6/TREMLl ECD vaccine. Data are shown as S/N ratio at the final serum dilution of 1 : 10000.

[0018] FIG. 7 shows blocking effects of anti-sera from RBD/TREMLl ECD vaccine or RBD/TREMLl ECD plus S2’-6/TREMLl ECD vaccine on RBD-ACE2 bindings at the final serum dilution of 1 :2000. Data are shown as mean ± SD. The dotted lines represent the cutoff at 20% inhibition.

[0019] FIG. 8 shows inhibitory effects of anti-sera from RBD/TREMLl ECD vaccine or RBD/TREMLl ECD plus S2’-6/TREMLl ECD vaccine on virus entry into host cells mediated by S-ACE2 interaction.

DETAIELD DESCRIPTION

[0020] Embodiments of the invention relate to SARS-CoV-2 vaccines using the S2’ peptides derived from the spike protein of SARS-CoV-2. These vaccines comprise S2’ peptides in unique nanocomplexes (NCs) that can elicit effective immune responses. Alternatively, the S2’ peptides are formulated with the TREMLl extracellular domain (ECD) or stalk peptide as immune boosters. TREMLl is triggering receptor expressed on myeloid cell-like transcript 1. As noted above, the S2’ peptides are important for viral entry into the host cells and the fusion peptides contained in the S2’ peptides are highly conserved among coronaviruses. These facts make the S2’ peptides ideal immunogens for designing SARS-CoV-2 vaccines. In addition, embodiments of the invention also relate to the use of the S2’ peptides as agonist adjuvants or immune boosters in other vaccines.

[0021] Inventors of the invention found an electro-kinetic approach to preparing nanoparticle- based vaccine. This approach is quite different from the conventional vaccine technologies. This technique manipulates the electric double layers of solution systems to encapsulate proteins with (+/-)-charged polymers by compressive force to form a stable, narrow charge-distribution, and dispersive spherical nanocomplexes (cf. U.S. Patent No. 10,052,390 B2; ELI: 2754436; China: CN103910892B; Taiwan: 1511744; the disclosures of all these patents are incorporated by reference in their entirety). Embodiments of the invention combine the nanoparticle-based approach with the SARS-CoV-2 S2’ peptide to achieve highly effective vaccines.

[0022] In accordance with some embodiments of the invention, the S2’ peptides may be further formulated with a unique immune booster. Inventors of the present invention have unexpectedly found that the extracellular domain (ECD) or its stalk of triggering receptor expressed on myeloid cell-like transcript 1 (TREMLl) can bind to TLR4/MD2 (LY-96, lymphocyte antigen 96). As a result of TREMLl ECD or its stalk peptide binding to the TLR4 complex, TREMLl ECD or its stalk can induce dendritic cell activation and maturation. Therefore, TREMLl ECD or its stalk can serve as vaccine adjuvants or immune boosters, as described in PCT/US2021/032620, filed on May 14, 2021, the disclosure of which is incorporated by reference in its entirety.

Selection of S2’ peptide sequence

[0023] A conserved genome that rarely mutates is very likely associated with vital functions of a virus. Some of the conversed genomes may play a critical part in the strategies that viruses use to enter host cells, especially at the catch-and-fuse process. As shown in FIG. 2, the S2’ cleavage site is highly conserved between the SARS-CoV and SARS-CoV-2. Studies showed that monoclonal antibodies which recognized this cleavage site inhibited propagation of SARS- CoV in monkeys (The Journal of Infectious Diseases 203(11): 1574-81). We searched databases for the genetic variation of SARS-CoV-2 and determined several conserved S2’ sequences that have especially been mapped against the antibodies from those convalescent SARS patients. These candidates were then screened and selected if any of them have been found to match the results of previous SARS vaccines in human and large animal studied. Some of the immunogen candidates we designed also possess overlapping sequences predicted to be a good immunogen against SARS-CoV-2 by bioinformatics analysis. The selected sequences are listed in Table I. These peptides have been tested as vaccines of the invention. The designed S2’ peptides have also been tested for the potencies in generating anti-viral antibodies in healthy animals. As used herein, the term “S2’ peptide” refers to a peptide derived from the sequence around the S2’ protease cleavage site in the spike protein of SARS-CoV-2, as illustrated in FIG. 2. S2’-l to S2’- 10 peptides refer to specific sequences listed in Table I.

Table I. S2’ peptide sequences

Generation of anti-sera against SARS-CoV-2 using the nanocomplex platform and TREMLl adjuvant platform.

[0024] The abilities of these S2’ peptides to induce immune responses were investigated by examining their impacts on the production of antibodies. Two different vaccine platforms were used. The first platform involves special nanocomplexes (NCs) to encapsulate the immunogens, and the second platform involves the use of a special immune response booster (i.e., TREMLl ECD or its stalk).

[0025] With the nanocomplex platform, immunogen peptides (e.g., the S2’ peptides) are encapsulated in nanocomplexes using a simple electro-kinetic approach by addition of a charged polymer solution into another oppositely charged polymer solution. For example, the immunogen peptides were mixed with poly-y-glutamic acid (g-PGA) (M.W. preferably about 200 kDa or less, e.g., 10-200 kDa, 50-200 kDa, or 100-200 kDa) to form a first charged polymer solution. Then, this solution was mixed with a second charged polymer solution (e.g., chitosan, CS) in an appropriate ratio. The molecular weight (MW) of chitosan is preferably about 10-100 kDa, adapted for adequate solubility at a pH value (e.g., pH value 5-9, preferably 6-8, more preferably 6.5-7.5) that maintains the bioactivity of protein and peptide drugs. The molecular weight (MW) of g-PGA or chitosan (CS) herein refers to the weight-averaged MW.

[0026] The exemplary ranges of concentrations for the immunogen peptides (e.g., S2’ peptides) and various components in the nanocomplexes are as follows: immunogen peptide (e.g., S2’ peptides): 0.5 to 2 mg/ml, chitosan (CS): 20 to 30 mg/ml, and g-PGA: 5-20 mg/ml. The resulting S2’ peptide-nanocomplexes (S2’ peptide/NCs) may be characterized with dynamic light scattering (DLS). The nanocomplexes (NCs) preferably have zeta potentials of from about +30 mV to about +50 mV and a size range preferably from about 100 nm to about 800 nm. These S2’ peptide/NCs are positively charged on the nanoparticle surfaces and are shown to have unusual abilities to induce immune responses, thereby having unusual therapeutic efficacies in the prevention and treatment of SARS-CoV-2 infections.

[0027] These S2’ peptide/NCs were tested for their abilities to elicit immune responses in BALB/c mice. FIG. 3 shows a schematic illustrating a test protocol. Briefly, S2’ peptide/NCs at 10 pg per dose, or NCs alone as a control, were inoculated into BALB/c mice through subcutaneous (sc) or oral route at day 0, 14, and 28. The blood samples were obtained before vaccination and at day 42.

[0028] The above embodiments use nanocomplexes as a vaccine platform. The nanocomplexes vaccines induce exceptionally robust immune responses. In addition, some embodiments of the invention use a second platform. The second vaccine platform involves the use of the extracellular domain (ECD) of TREM-like transcript- 1 (TREML1) protein. TREML1 comprises an ECD (residues 1-162 or 16-162), a transmembrane domain (residues 163-183), and a cytoplasmic tail (residues 184-311). The ECD of TREML1 comprises a single immunoglobulin variable (IgV) domain (residues 16-121) and a stalk (residues 122-162). In accordance with embodiments of the invention, the TREML1 ECD may be a mature ECD (residues 16-162) or a pro-ECD (residues 1-162, including the signal peptide), both of which will be generically referred to herein as TREML1 ECD. Embodiments of the invention may use a TREML1 ECD or its stalk from human or another mammal (e.g., mouse). Human TREML1 consists of 158 amino acids and has a molecular mass of 17.3 kDa.

[0029] TREML1 is exclusively found on platelets in the peripheral blood of humans. Upon platelet activation, TREML1 is quickly exposed on the membrane and subsequently cleaved, leading to the release of a soluble fragment (sTREMLl). Prior studies suggest that soluble TREML1 plays an important role in the inflammatory related disease. We found that soluble TREML1 can directly bound to monocytes and modulate immune responses (WO2016197975A1, the disclosure of which is incorporated by reference herein) and that TREML1 ECD or its stalk can bind to TLR4/MD2 (LY-96, lymphocyte antigen 96). As a result of TREML1 ECD or its stalk binding to the various TLRs, TREMLl ECD or its stalk can induce dendritic cell activation and maturation. Therefore, TREMLl ECD or its stalk can serve as vaccine adjuvants or immune boosters, as described in PCT/US2021/032620, filed on May 14, 2021, the disclosure of which is incorporated by reference in its entirety.

[0030] In accordance with embodiments of the invention, TREMLl ECD or TREMLl stalk peptides can function as immune boosters. S2’ peptides may be mixed with recombinant or synthetic TREMLl ECD protein or a TREMLl stalk peptide in an appropriate ratio. Examples of TREMLl ECD and stalk peptide sequences are shown in Table II. These vaccines may be referred to as TREMLl adjuvant vaccines in this description. Each vaccine formulation may have an appropriate ratio of the S2’ peptide and the TREMLl ECD or stalk, e.g., 1-100 pg (preferably about 50 pg) S2’ peptide and 1-50 pg (preferably about 20 pg) recombinant or synthetic TREMLl ECD protein or stalk peptide in a typical vaccine dose.

Table II: TREMLl ECD or Stalk Peptides used in exemplary embodiments of the invention.

[0031] To test the abilities of these vaccines to induce SARS-CoV-2 antibodies, BALB/c mice were immunized subcutaneously (sc) with a TREMLl adjuvant vaccine (e.g., 50pg S2’ peptide and 20pg recombinant TREMLl ECD protein), and then booster vaccinations were administered subcutaneously using dosages similar to those for primary immunization on day 14 and day 28 post-primary immunization (as illustrated in FIG. 3). TREMLl ECD alone (without S2’ peptide) was used as a control.

[0032] To determine anti-S2’ peptide antibody levels in the vaccinated mice, antisera were collected on day 42 post-immunization. ELISA was performed using S2’ peptides. To assess the polarization/activation of T helper (Th) cells, IgG2a and IgGl immunoglobulin isotypes are used as markers for Thl and Th2 lymphocytes, respectively. As shown in FIG. 4, the S2 ^, -6/TREMLl ECD vaccine induced very high levels of serum S2’ -6-specific IgGl in mice, whereas TREMLl ECD alone did not. IgG2a titers were also significantly induced by the S2’-6/TREMLl ECD vaccine, but not by the TREMLl ECD alone. Similar results were observed in the mice treated with S2’-l/NCs vaccine. S2’-l/NCs induced both high titers of S2’-l-specific IgGl and IgG2a, whereas NCs alone did not. These results show that S2’ peptide vaccines of the invention have properties that can polarize T helper cells to induce both Thl and Th2 functions, suggesting that these vaccines can induce both cellular immune responses and humoral immune responses. [0033] We next examine whether S2’ peptide vaccines can generate antibodies that can bind to the tertiary structure of the spike protein (i.e., the native structure of the spike protein). SARS- CoV-2 spike extracellular domain (ECD) was used for ELISA assay. As shown in FIG. 5, mouse sera from BALB/c mice after S2’-6/TREMLl ECD and S2’-l/NCs vaccinations were able to generate high titers of anti-SARS-CoV-2 spike (ECD) antibodies, while those vaccinated with TREML1 ECD alone or nanocomplexes alone did not. In conclusion, S2’ peptide vaccinations not only can generate high titers of antibodies that can bind to primary structure of S2’ peptide but also can recognize tertiary structure of spike protein.

S2’ peptide vaccine can be used as an agonist adjuvant to induce strong neutralization antibody responses

[0034] Many peptides such as tetanus toxin peptide (TT peptide) or pan HLA DR-binding epitope (PADRE) can activate antigen specific-CD4+ T cells (U.S. Patent No. 9,249,187 B2). Thus, these peptides can be used as an agonist adjuvant in vaccine developments. Compared with RBD/TREMLl ECD vaccine, it was surprisingly found that mouse sera from BALB/c mice after RBD/TREMLl ECD+S2’-6/TREMLl ECD vaccination were able to generate higher titers of anti-SARS-CoV-2 RBD antibodies (FIG. 6) than that induced by RBD/TREMLl ECD vaccine. These results indicated that S2’ peptide vaccine could strongly enhance anti-RBD antibody response when combine with RBD-based vaccine. This is probably due to the fact that S2’ peptide vaccines can polarize T helper cells to induce both Thl and Th2 functions, which then enhance the immune responses of the RBD/TREMLl ECD vaccine.

[0035] The antisera were also tested for their blocking effects against RBD-ACE2 binding. As shown in FIG. 7, mouse sera from BALB/c mice after RBD/TREMLl ECD vaccination generated antibodies that can block RBD-ACE2 interactions in vitro. Compared with RBD/TREMLl ECD vaccination, RBD/TREMLl ECD combined with S2’-6/TREMLl ECD vaccination generated substantially higher titers of blocking antibodies to inhibit RBD-ACE2 interactions. The mean inhibition rates for RBD/TREMLl ECD alone and RBD/TREMLl ECD+S2’-6/TREMLl ECD vaccines, respectively, were 29.97% and 53.31% at a final serum dilution of 1:2000.

[0036] The antisera were also tested for their neutralization capacity against SARS-CoV-2 infection using a pseudovirus neutralization assay. The neutralization assays were performed by incubating pseudovirus with serial dilutions of serum, HEK-293T cells stably expressing human ACE2, and TMPRSS2 genes to see if the vaccinated mouse serum could protect cells from virus infections. As shown in FIG. 8, mouse sera from BALB/c mice after RBD/TREMLl ECD vaccination generated high titers of neutralizing antibodies and prevented pesudovirus infection in vitro. Compared with RBD/TREMLl ECD vaccination, RBD/TREMLl ECD combined with S2’-6/TREMLl ECD vaccination generated higher titers of neutralizing antibodies to prevent pseudovirus infection. The IC50 values (based on serum dilutions) for RBD/TREMLl ECD vaccine and RBD/TREMLl ECD+S2’-6/TREMLl ECD vaccine were 243.4 and 529.7, respectively. In conclusion, S2’ peptide vaccine helps RBD-based vaccine to induce a significantly stronger neutralization antibody response. Thus, in addition to functioning as an immunogen, S2’ peptides can also serve as agonist adjuvants or carrier epitopes suitable for use in the development of vaccines.

[0037] Embodiments of the invention will be further illustrated with following specific examples. One skilled in the art would appreciate that these specific examples are for illustration only and that other modifications and variations are possible without departing from the scope of the invention.

EXAMPLES

Preparation and Characterization of S2’-l/NCs.

[0038] A first solution is prepared with g-Polyglutamic acid (g-PGA; w/v=l% in ddtTO; weight-averaged M.W. range = about 200 kDa or less, such as about 150-200 kDa) and a predetermined amount of S2’-l peptide. A second solution is prepared with chitosan in 1% acetic acid (w/v=2.5% chitosan, weight averaged M.W. range = about 10-100 kDa). Add the second solution (chitosan solution) to the first solution (y-PGA with S2’-l peptide) to form nanocomplexes (NCs). NCs were stored at 4°C overnight for the stability tests. The sizes, zeta- potentials, and polydispersity index (Pdl) were determined with Malvern Zetasizer Nano Series (Zetasizer Nano ZS, Malvern Panalytical Ltd., U.K.).

Preparation of RBD/TREMLl ECD and S2’-6/TREMLl ECD [0039] The antigen solution is prepared with 20 pg TREMLl ECD and various antigens (example: 20pg RBD or 50 pg S2’-6 peptide) in 50pl PBS. Add Alhydrogel® adjuvant 2% (InvivoGen, San Diego, CA, USA) to the antigen solution; the final volume ratio of Alhydrogel® adjuvant 2% to antigen solution was 1:1. Mix well by pipetting up and down for 5 minutes to allow Alhydrogel® adjuvant 2% to effectively adsorb the antigen and TREMLl ECD. The vaccine may be stored at room temperature.

Antibody induction experiments

[0040] All animal studies were conducted under specific pathogen-free conditions. In antibody induction experiments, six- to eight-week-old female BALB/c mice were divided into 2 groups: NC only and 10 pg/dose S2’-1/NC. Mice were inoculated with these vaccines at days 0, 14, and 28. The mouse blood samples were collected before vaccination and at day 42. In related studies, six- to eight-week-old male BALB/c mice were divided into 4 groups: TREML1 ECD only, 20 pg/dose RBD/TREMLl ECD, 50 pg/dose of S2’-6/TREMLl ECD, or 20 pg/dose RBD/TREMLl ECD + 50 pg/dose of S2’-6/TREMLl ECD. Mice were inoculated with these vaccines at days 0, 14, and 28. The mouse blood samples were collected before vaccination and at day 42.

Inoculation route

[0041] Vaccines of the invention may be administered via any suitable routes, such as subcutaneous (sc), intravenous (iv), intramuscular (im), intraperitoneal (ip) injections, as well as via oral route or nasal route.

Enzyme-linked immunosorbent assay (ELISA) for anti-S2’-l, anti-S2’-6, anti-RBD, and anti spike antibodies detection.

[0042] SARS-CoV-2 S2’-related peptide, RBD, or extracellular domain of spike (Spike ECD, GenScript) proteins are coated on 96-well plates at 5 ug/ml and kept at 4°C overnight. Mouse sera are serially diluted and added into each well to incubate for 2 hours at room temperature. Anti-mouse IgGl or anti-mouse IgG2a antibodies or anti-mouse IgG conjugated with HRP are added into wells and incubated for 30 min at room temperature. After wash, TMB substrate was added to produce color products, and the reactions were stopped by addition of IN HC1. The optical density of each well was determined immediately, using a spectrophotometer set to 450 nm and 540 nm. The results are shown in FIG. 4-6.

SARS-CoV-2 surrogate virus neutralization assay

[0043] Neutralization assays of antibody-mediated blockage of ACE2-RBD protein-protein interactions were performed by SARS-CoV-2 Surrogate Virus Neutralization Test Kit purchased from GenScript (Piscataway, NJ, USA). The results are shown in FIG. 7.

Pseudotyped virus neutralization assay

[0044] Neutralization assays were performed by incubating pseudovirus with serial dilutions of heat inactivated serum, HEK-293T cells (1 ^c 10 ⁴ ) stably expressing human ACE2 and TMPRSS2 genes in 50 pL of opti-MEM (Gibco) were seeded in each well of a black pCLEAR flat-bottom 96-well plate (Greiner Bio-one™). The cells were incubated overnight at 37°C with 5% CO2. On the following day, each serum was twofold serially diluted in opti-MEM and incubated with SARS-CoV-2 pseudotyped lentivirus at 37°C for 1 h. The virus-serum mixture was transferred to the 293T/ACE2/TMPRSS2 cell plate with the final multiplicity of infection (MOI) of 0.1. For each serum, the starting dilution was 1/10 with seven twofold dilutions to the final dilution of 1/640. The culture medium was then replaced with fresh DMEM (supplemented with 10% FBS, 100 U/ml Penicillin/Streptomycin) at 16 hr post-infection, and cells were continuously cultured for another 56 hr. After incubating the infected cells at 37°C for 72 h, cells were quantified for GFP fluorescence on ImageXpress Micro Confocal High Content Imaging System (Molecular Devices).

Image and statistical analysis

[0045] After incubating the infected cells at 37°C for 72 h, the cells were stained with DAPI at 37°C for 20 min, and then GFP-positive cell and total cell nucleus were detected on ImageXpress Micro Confocal High Content Imaging System (Molecular Devices). The raw images (5 x 5 sites, total 25 sites) were acquired using 20 ^c water immersion objective lens, processed, and stitched using the appropriate setting. The total cells (indicated by nucleus staining) and GFP positive cells were quantified for each well. All analysis were carried out using MetaXpress Cell Scoring module, counting positive cell and total cell number at each site. After Cell Scoring analysis, the raw data was processed by Lumi-Vcal (LumiSTAR customized analysis software). Transduction rates were determined by dividing the GFP-positive cell number with the total cell number. Relative transduction rates were obtained by normalizing the infection rates of serum treated groups to those of non-serum-treated controls. Inhibition percentage was obtained by considering the non-serum-treated control as 0% inhibition. The curves of the relative inhibition rates versus the serum dilutions were plotted using Prism 8 (GraphPad). A nonlinear regression method was used to determine the dilution fold at which 50% of GFP fluorescence (IC50) was expressed. Each serum was tested in triplicates. All SARS- CoV-2 pseudovirus neutralization assay was performed at the BSL-2 facility. Results are shown in FIG. 8.

[0046] Embodiments of the invention have been described with a limited number of examples. These examples are for illustration only and are not meant to limit the scope of the invention. One skilled in the art would appreciate that other modifications and variations are possible without departing from the scope of the invention. Therefore, the scope of protection should only be limited by the attached claims.