OF CORONAVIRUS INFECTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Application No. 63/307,579, filed on February 7, 2022, and U.S. Provisional Application No. 63/380,585, filed on October 24, 2022 the content of which is incorporated herein in its entirety by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created February 7, 2023, is named "OBIP-8PCT_SEQ.List_CT26.xml" and is 3,217 bytes in size.

FIELD

[0003] The present disclosure relates to a COVID-19 vaccine that can provide protection against a broad spectrum of SARS-CoV-2 variants. The disclosure provides a COVID-19 vaccine (OBI- BCVax) that can provide protection against a broad spectrum of SARS-CoV-2 variants and spike protein of delta strain is combined with QS-21 based immune stimulating complex (AB801- ISCOM and OBI821-ISCOM) adjuvant.

BACKGROUN D OF THE INVENTION

[0001] Since the outbreak of SARS-CoV-2 pandemic occurred in Dec. 2019, there are over 615 million confirmed cases and 6.5 million deaths of the world, as of 2022 Oct. The persistence and wide spread of this pandemic greatly impact global economy and public health. Tremendous endeavors have been taken to develop vaccines to control the pandemic. Up to date, as many as 34 vaccines have been authorized as either a primary vaccination or booster (Polack FP et al., 2020; Baden LR et al., 2021; Falsey AR et al., 2021; Stephenson KE et al., 2021). However, constantly evolution of the virus renders the emergence of several variants in less than two years such as alpha, beta, gamma, delta, and the current dominant omicron strains (Hadj Hassine I, 2022; Thakur V and Ratho RK, 2022). Given that most of the COVID-19 vaccines were developed based on wild type strain, the emerged variants are capable of escaping the induced immune responses compromising the protectivity of the vaccines. Lopez Bernal reported that the effectiveness of vaccine after one dose was notably lower against delta variant compared to alpha variant infection (Lopez Bernal et al., 2021). Several studies indicated that, after two doses of vaccination, the serum neutralization titer against delta strain was significantly reduced compared to wild type, and significantly lowered neutralization activity was detected against Omicron strain (Schubert M et al., 2022; Andrews N et al., 2022; Smid M et al., 2022). These observations clearly demonstrated that the protectivity of current vaccines may not be sufficient for newly emerged variants. A second-generation vaccine targeting variant strain may be an effective way to enhance the protectivity. In this study, we aim to develop a second- generation vaccine that is capable of inducing immunity against variants including delta and omicron strains either as a primary vaccination or a booster.

[0002] Updates on SARS-CoV-2 classifications, the geographic distribution of Variants of Concerns (VOCs), and summaries of their phenotypic characteristics (transmissibility, disease severity, risk of reinfection, and impacts on diagnostics and vaccine performance) based on published studies, are regularly provided in the WHO Weekly Epidemiological Updates (https://www.who.int/en/activities/tracking-SARS-CoV-2-varia nts/). Currently WHO label included: Alpha, Beta, Gamma, Delta, and Omicron strains.

SUMMARY OF THE INVENTION

[0003] The present disclosure provides an immunogen, which including an engineered coronavirus spike protein having an amino acid sequence of SEQ ID No: 1, a variant having at least 90% sequence identity to the amino acid sequence of SEQ ID No: 1 or an immunologically active fragment.

[0004] In some embodiments, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0005] In some embodiments, the variant is the D614G, alpha, beta, gamma, delta, lambda, omicron strain or a combination.

[0006] In some embodiments, the variant is the delta strain.

[0007] In some embodiments, the variant is the omicron strain.

[0008] In some embodiments, the engineered coronavirus spike protein is trimer format. [0009] In some embodiments, the engineered coronavirus spike protein is capable of eliciting an enhanced immune response relative to the native coronavirus spike protein.

[0010] In some embodiments, the enhanced immune response is an increased IgM titer, an increased IgG titer or an increased neutralization titer.

[0011] The present disclosure also provides a vaccine including immunogen and adjuvant. Further, the immunogen including an engineered coronavirus spike protein having an amino acid sequence of SEQ ID No: 1, a variant having at least 90% sequence identity to the amino acid sequence of SEQ. ID No: 1 or an immunologically active fragment.

[0012] In some embodiments, the adjuvant is selected from the group consisting of aluminum hydroxide, Freund's adjuvant, CpG adjuvant, QS-21 adjuvant, AB801 adjuvant or AB801-ISCOM adjuvant.

[0013] The present disclosure provides a COVID-19 vaccine that can provide protection against SARS-CoV-2 variants, especially the delta and omicron variants. The vaccine can be employed as a protectivity enhancement shot to subjects who have been vaccinated with any type of vaccines against mutant variants.

[0014] The present disclosure further provides a method for preventing or enhancing immunity of a subject against coronavirus infection. The method includes administering to a patient subject in need thereof an effective amount of immunogen or vaccine.

BRIEF DESCRIPTION OF DRAWINGS

[0015] A complete understanding of the invention may be obtained by referencing the accompanying drawings when considered in conjunction with the subsequent detailed description. The embodiments illustrated in the drawings are intended only to exemplify the invention and should not be construed as limiting the invention to the illustrated embodiments.

[0016] Figure 1. Linear diagram of the delta-S spike protein sequence. The mutation sites of delta strain including T19R, G142D, A156-157, R158G, L452R, T478K, D614G, P681R, and D950N were as indicated.

[0017] Figure 2. SDS-PAGE analysis with Instant Blue staining of delta-S spike protein under denaturing conditions.

[0018] Figure 3. Characterization of delta-S spike protein. Characterization of spike protein candidates by SEC-UV analysis.

[0019] Figure 4. Binding activity between delta-S spike protein and human ACE2. [0020] Figures 5. Characterization of the size of AB801-ISCOM and the transmission electron microscopy (TEM) Image of AB801-ISCOM and delta S protein trimer. Figure 5A, Dynamic light scattering (DLS) analysis of AB801-ISCOM. Duplicate analysis was performed to demonstrate the consistency. Figure 5B, Transmission electron microscopy (TEM) Image of AB801-ISCOM. Figure 5C, The negative stain EM structural analysis of OBI-BCVax, consisting of delta S protein and AB801-ISCOM. The EM image showed delta S protein in trimer structure with homogenous distribution. The size of delta S protein is about 20 nm, and AB801-ISCOM is about 40 nm. Figure 5D, The LC-MS chromatogram of AB801 adjuvant.

[0021] Figures 6. Evaluation of the immunogenicity of OBI-BCVax in BALB/c mice. Figure 6A, Treatment and sampling schedule. The delta S protein (10 pg) combined with or without adjuvant candidates of aluminum phosphate plus CpG 1018 (50 pg plus 10 pg, triangle), AB801 (5 or 10 pg, square), or AB801-ISCOM (5 or 10 pg, circle, grey bars) were immunized to BALB/c mice on day 0 and day 14. Figure 6B, anti-S protein IgG titer of wild type, delta, and omicron strains in immunized mouse serum collected on day 28. Figure 6C, Neutralizing antibody titers of sera of immunized mice against SARS-CoV-2 pseudoviruses D614G, B.1.1.7, 507Y.V2, P.l, B.1.617.2, BA.l, and BA.2 variants. Bars indicate the geographic mean titer (GMT), and the error bars represent 95% confidence intervals. Data were analyzed using one-way ANOVA. Figure 6D, pNT50 value of AB801-ISCOM groups against D614G, B.l.1.7, 507Y.V2, P.l, B.l.617.2, BA.l, and BA.2 variants. (*p <0.05)

[0022] Figures 7. T cell responses in OBI-BCVax immunized BALB/c mice. The delta S protein (10 pg) combined with or without adjuvant candidates of aluminum phosphate plus CpG 1018 (50 pg plus 10 pg, triangle), AB801 (5 or 10 pg, square), or AB801-ISCOM (5 or 10 pg, circle, grey bars) were immunized to BALB/c mice on day 0 and day 14, splenocytes were harvested on day 28 for analysis. Figure 7A, Flow cytometry analysis of CD4 T cell populations of CD4 ⁺ IFN-y ⁺ and CD4 ⁺ IL-4 ⁺ cells. Figure 7B, CD8 T cell populations of CD8 ⁺ IFN-y ⁺ and CD8+Granzyme B+ cells were evaluated using immunized mice. Figure 7C, The number of IL-2, IFN-y, and IL-4 secreting cells from immunized splenocytes per 2.5E+5 cells were analyzed by ELISPOT assay. Figure 7D, The ratio of I FN-y/l L-4 of ELISPOT results were calculated. Bars represent mean ± SD. Statistically significant differences were indicated (*p < 0.05).

[0023] Figures 8. Evaluation of the immunogenicity of OBI-BCVax candidates in BALB/c mice with booster injection. Group of mice were immunized with 10 pg of delta S protein combined with different dosage of AB801-ISCOM (7.5, 5, or 2 pg) on day 0 and day 14 (white bars). Another group of mice were received first two injections of 10 pg delta S protein with 7.5 pg AB801-ISCOM on day 0 and day 14, and a booster of same dosage on day 56 (gray bars). Figure 8A, The treatment schedule and sampling time. Figure 8B, anti-S protein IgG titers of delta, omicron BA.2 and BA.5 strains in immunized mouse serum collected on day 84. The time course of serum IgG response is illustrated in Figure 8C. Figure 8D, pseudovirus neutralizing antibody titers of sera collected at day 84 of immunized mice against SARS-CoV-2 pseudoviruses B.1.617.2, BA.2, and BA.4/BA.5 variants. Bars indicate the GMT, and the error bars represent 95% confidence intervals. Data were analyzed using one-way ANOVA. Figure 8E, live virus neutralizing antibody titers of sera collected at day 84 of immunized mice against SARS-CoV-2 live viruses B.1.617.2 and BA.l variants. Figure 8F, CD8 T cell populations of CD8 ⁺ IFN-y ⁺ and CD8 ⁺ Granzyme B ⁺ cells were evaluated using immunized mice splenocytes harvested on day 84. Bars represent mean ±SD. Statistically significant differences were indicated (*p < 0.05).

DETAILED DESCRIPTION OF TH E I NVENTION

[0024] As used herein, the articles "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0025] An "effective amount," as used herein, refers to a dose of the vaccine or pharmaceutical composition that is sufficient to reduce the symptoms and signs of coronavirus infection.

[0026] The term "subject" can refer to a vertebrate having cancer or to a vertebrate deemed to be in need of cancer treatment. Subjects include all warm-blooded animals, such as mammals, such as a primate, and, more preferably, a human. Non-human primates are subjects as well. The term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary uses and medical formulations are contemplated herein.

[0027] The spike protein (S protein) on the SARS-CoV-2 virus surface plays an important role in facilitating viral infection via the binding to ACE2 receptor of host cells. This makes the S protein a prime antigen to be targeted for vaccine development, which could induce neutralizing antibodies that blocks viral infection.

[0028] Different variants of SARS-CoV-2 have emerged in UK, Brazil, California, India, south Africa and other areas of the world. Delta variant B.1.617.2 (India variant) and omicron variant (B.1.1.529) were found to increase the transmissibility and reinfection rate. Current existing vaccines show reduced neutralizing activity against the variants, especially the delta and omicron strain, which may increase the reinfection rate rendering further mutation and spread of the pandemic. Given the resurgence of infection in several countries despite high vaccination rate, there is an urgent need to develop a vaccine that is capable of neutralizing such variants.

[0029] We aim at developing such a vaccine by using spike protein of delta strain as the antigen to provide more comprehensive protection against mutation strains. Here we developed a COVID-19 vaccine (OBI-BCVax) using delta strain spike protein as the antigen to provide a more comprehensive protection against delta strain, and possibly certain degree of protection against other known variants. In combination with a saponin based adjuvant ISCOM (immune stimulating complex) in nanoparticle format, which provides better immunogenicity towards T cell responses than traditional Q.S-21.

[0030] In an embodiment the substantially cage-like nanoparticles have a particle size of between 10-5000 nm, both values as measured by dynamic light scattering (DLS).

[0031] In an embodiment of the substantially cage-like nanoparticles, the ratio between saponin and cholesterol is from 1:1 to 10:1; and the ratio between DOPC (1,2-dioleoyl-sn- glycero-3-phosphocholine) and cholesterol is from 1:1 to 10:1.

[0032] In another embodiment the substantially cage-like nanoparticles, the ratio between AB801, cholesterol and DOPC (l,2-dioleoyl-sn-glycero-3-phosphocholine) is from 1:1:1 to 10:1:10, preferably 5.2:1:2.

[0033] Based on the concept mentioned above, the current study evaluates the immunogenicity of delta strain SARS-CoV-2 spike protein in combination with AB801-ISCOM adjuvant to identify a suitable fit COVID-19 vaccine (OBI-BCVax) candidate for further development.

[0034] EXAMPLES

[0035] Example 1: SARS-CoV-2 virus spike protein preparation and characterization

[0036] To produce SARS-CoV-2 spike protein, a plasmid containing delta strain spike protein sequence were transduced to ExpiCHO-S cells (Thermo Fisher Scientific; Cat. No.A29127). The design of protein sequence for delta S protein production is shown in Figure 1. A human rhinovirus 3C protease (HRV3C) recognition sequence was inserted into the sequences between spike protein and His tag to facilitate the removal of His tag after purification. The DNA was then ligated into pcDNA3.4 expression vector (Thermo Fisher) using NEBuilder DNA Assembly kit (Azenta Life Sciences). The delta strain SARS-CoV-2 Spike protein contain T19R, G142D, EF156- 157del, R158G, L452R, T478K, D614G, P681R, D950N mutations compared to wild type SARS- CoV-2 spike protein sequence, the mutation sites were consistent with the identified B.1.617.2 variant. The culture supernatants were collected at 8-11 days post-transfection, depending on cell viability. The cell culture supernatant containing spike protein was purified by Ni column (HisPur™ Ni-NTA Resin; Thermo Fisher Scientific; Cat. No.88821). The eluted sample was concentrated by Amicon® Ultra-15 Centrifugal Filter Unit (Merck KgaA; Cat. No.UFC9100) and stored in 4 °C. The purified delta-S spike protein was verified by 12% SDS-PAGE, as shown in Figure 2. Under denaturing conditions, the S protein showed a major band at 140 KDa, which matches to the molecular weight of monomer S protein.

[0037] SEC-SUV analysis

[0038] A total of 10 pg sample was analyzed per injection, SARS-CoV-2 spike protein was used as the control sample (SST). SRT SEC-500, 7.8x300 mm column (Sepax Technologies; Cat. No.215500-7830) was applied with mobile phase 150 mM PBS pH 7.0. Isocratic elution at 0.5 mL/min and acquisition time at 30 min. Figure 3 showed the chromatogram profile of purified delta strain of SARS-CoV-2 spike protein under the analysis by SEC column with UV detection. The analysis result indicates 86-90% in trimer format, and 9-13% high molecular weight species (%HMWS) with or without His tag removal.

[0039] Example 2: SARS-CoV-2 spike protein and human ACE2 (Angiotensin-converting enzyme 2) binding activity

[0040] To determine the binding kinetics of human ACE2 for SARS-CoV-2 spike protein, biolayer interferometry (BLI) experiments were performed on an Octet instrument (ForteBio). ACE2 protein with Fc tag was first captured on Anti-hlgG Fc Capture (AHC) biosensors. SARS-CoV-2 spike proteins were diluted with assay buffer (0.1% BSA in PBS, 0.05% Tween 20). The final concentrations of S protein were 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 nM. The results were shown in Figure 4. The association and dissociation constant of ACE2 with S protein was performed in assay buffer at the indicated concentration for 600 seconds and 900 seconds, respectively. The on-rate and off-rate were determined to be 3.45x105 (M-1S-1) and 1.11x10-4 (S-l), and the KD value was calculated using a 1:1 global fit model using ForteBio's Data Analysis software. Figure 4 indicated that the KD of SARS-CoV-2 spike protein is indicated as 3.22E-10 (0.32 nM). The binding affinity was ten-fold higher than the findings reported by other study groups with delta strain S protein (KD= 41 nM) (Lan J et al., 2020; Walls AC et al., 2020; Wang Y et al., 2022), potentially due to the lower off-rate detected in our delta S protein.

[0041] Example 3: ISCOM adjuvant preparation

[0042] Adjuvant OBI821 is a saponin-based adjuvant derived from the bark of the Quillaja Saponaria (Q.S) Molina tree. Adjuvant OBI821 is structurally similar to Q.S-21 adjuvant based on the comparison of physicochemical data. Both OBI821 and Q.S-21 exist as mixtures of isomers. There are total of six isomers found in OBI821 adjuvant substance (AS). These six isomers can be classified into three groups, 1990-V1, 1990-V2 and 1858. Each group contains two regioisomers that can be identified as group A and B. The detail of OBI821 adjuvant is as disclosed in PCT publication number: WO2019/191317.

[0043] Adjuvant AB801 is also a saponin-based adjuvant derived from the bark of the Quillaja Saponaria (QS) Molina tree. Adjuvant AB821 is structurally similar to OBI-821 adjuvant based on the comparison of physicochemical data. However, there are total of seven isomers found in AB801 adjuvant substance (AS). The chemical structure of AB801 is listed in the following Formula (I):

[0044] The seven isomers are listed in Table 1. The LC-MS chromatogram of AB801 was showed in Figure 5D. It indicated the major isomers of AB801 are AB801-1858 (Peak 1: 23.5%) and AB801-1990 (Peak 3: 67.1%). However, the AB801-1872 (Peak 2: 2.3%) and AB801-2004 (Peak 4: 4.8%) isomers only existed in AB801, not in OBI821.

[0045] Table 1. The seven isomers of AB801 adjuvant substance (AS)

[0046] AB801-ISCOM is consist of AB801 (Amaran), cholesterol (Sigma), and DOPC (1,2- dioleoyl-sn-glycero-3-phosphocholine) (Avanti). Cholesterol 2.32 mg and DOPC (1,2-dioleoyl-sn- glycero-3-phosphocholine) 4.72 mg were dissolved in 1.5 mL chloroform into a 10-mL round bottom flask. Chloroform was evaporated off on a rotary evaporator at a water bath temperature of 45±5 °C under 150 mbar vacuum and a lipid-film structure was formed. The lipid-film was hydrated with 9 mL TBS (Tris buffered Saline) buffer containing 12 mg AB801 and 120 mg P-OG (Octyl-beta-D-glucopyranoside) and the mixture was shaken for two hours at room temperature. The solution was transferred into a RC dialysis membrane with a molecular weight cut-off of 1 KDa. The samples were dialyzed at 4±2 °C against 8 liters of TBS buffer with stirring by SpectraFlo™ Dialysis System for 48 hours. After dialysis, the solution was sampled to test the P-OG residual and particle size (IPC). Finally, the solution was sterilized by filtration in a laminar flow.

[0047] Furthermore, we also tried different methods (e.g. lipid-film hydration, ethanol injection or reverse ethanol injection) for ISCOM-matrix preparation except dialysis method (Julia M et al., 2006). The characterization by dynamic light scattering (DLS) of OBI821-ISCOMs were listed in Table 2.

[0048] Table 2. The characterization by dynamic light scattering (DLS) of OBI821-ISCOMs

[0049] These components were mixed in 5.2:1:2 ratio followed by dialysis to form ISCOM nanoparticle matrix as previously described (Lendemans DG et al., 2006). In brief, phosphatidylcholine and cholesterol were dissolved in chloroform and then mixed in a glass vial. The resulting solution was transferred to a round bottom flask and the chloroform was evaporated in a rotary evaporator under vacuum followed by addition of AB801 and P-OG (Sigma) in Tris-buffered saline (TBS). The mixture was then transferred to a shaker and stirred at room temperature until an optically clear micellar solution was obtained. The solution was transferred to a dialysis bag with molecular weight cut-off of 1 KDa followed by dialyzing against IL TBS to remove excessive cholesterol or DOPC. Buffer exchange was performed every 8 to 16 hours for a period of 48 hours to give AB801-ISCOM with an average particle size of 40 nm.

[0050] The delta S protein was prepared in solution of 20 mM Tris (pH 8.0), 150 mM NaCI and AB801-ISCOM was prepared in solution of 145 mM TBS (pH 7.4). Delta S protein and ISCOM were mixed at a ratio of 3:2 followed by dilution to give final the concentrations of delta S protein (20 pg/mL) and AB801-ISCOM (13.3 pg/mL). The EM images were taken by negative stain method. A single drop of sample solution (4 pL) was deposited onto glow-discharged carbon-coated copper grid and washed with deionized water. After staining with 2% (w/v) uranyl acetate for 45 seconds, the solution was removed by filter paper and the grid was placed on bench to allow for air dry before taking images. Images were recorded using a JEM-1400 Transmission Electron Microscope (JEOL) equipped with a field emission electron source under an acceleration voltage of 120 keV at the IMANI center/NCKU (Tainan, Taiwan). Images were taken with a camera system (Model 895; Gatan, Inc. Ultrascan 40004K x 4K CCD) at 80,400x magnification and -0.75-1.81 pm nominal defocus.

[0051] The ISCOM was prepared by mixing AB801, cholesterol, and DOPC (dipalmitoylphosphatidylcholine) at 5.2:1:2 ratio followed by dialysis with 4 times of buffer exchanges. Homogenous ISCOM nanoparticles were achieved through the process of dialysis. The size of nanoparticle was determined to be 40 nm in average by dynamic light scattering (DLS) with polydispersity index (PDI) of 0.138 suggesting good monodispersity of the nanoparticles (Figure 5A).

[0052] Transmission electron microscopy (TEM) was employed to characterize the ISCOM structure. As shown in Figure 5B, a uniform and homogeneous cage-like structure was observed. When delta S protein was mixed with AB801-ISCOM, the majority of S protein was in singular trimer format under TEM as indicated in Figure 5C (upper right), which is consistent with the SEC analysis result. In addition, the cage-like structure of AB801-ISCOM was maintained in the presence of the S protein as shown in Figure 5C (lower right). The TEM results indicated that there was no interaction between cage-like ISCOM and S protein, and there was no aggregation detected.

[0053] Example 4: Animal study of COVID-19 vaccine (OBI-BCVax) candidates immunogenicity evaluation

[0054] 4-1: Immunogenicity of OBI-BCVax in mice

[0055] Seven weeks old female BALB/c mice (n=5 per group) were immunized with delta S protein (10 pg) in combination with different adjuvant candidates, including aluminum hydroxide (50 pg) plus CpG 1018 (10 pg), AB801 (5 or 10 pg), and AB801-ISCOM (5 or 10 pg) by intramuscular (IM) injection. For control group, mice were injected with S protein only (10 pg). All mice received two injections on day 0 and day 14. Mice were sacrificed on day 28 post first immunization and the spleen and serum of each mouse were collected for further analysis.

[0056] The anti-S protein ELISA was used to determine the mouse sera IgG titer. Briefly, SARS- CoV-2 S protein of different variants (Aero Biosystems) were coated on the 96-well microtiter plates at 4 °C overnight. After blocked with 1% BSA in PBS, the test samples were diluted in twofold of serial dilution with a dilution factor up to 1:250 followed by adding to the microtiter plates and incubated at 25 °C for 60 minutes. After being washed 3 times with PBST buffer, the goat anti-mouse IgG-HRP conjugate was added to the solution. The resulting mixture was incubated at 25 °C for 60 minutes followed by addition of substrate (3, 3', 5,5'- tetramethylbenzidine) and then stop solution (IN HCI). The absorbance of each well was read at 450 nm in a microtiter plate reader. SARS-CoV-2 variants including wild type, alpha (B.l.1.7), beta (B.1.351), gamma (P.l), delta (B.1.617.2), omicron (BA.l, BA.2, and BA.5) were employed for the evaluation. The titer of each sample was determined by the highest fold of dilution with OD value that was higher than cut-off point. [0057] Groups of BALB/c mice were immunized with delta S protein in combination with various adjuvants including aluminum hydroxide plus CpG 1018, AB801, and AB801-ISCOM. The immunization schedule is shown in Figure 6A. After two injections on day 0 and day 14, serum samples were harvested on day 28 for anti-S protein IgG titer evaluation. The IgG titers against wild type, delta (B.1.617.2), and omicron (BA.l and BA.2) strain S protein were shown in in Figure 6B. The results indicated that AB801-ISCOM adjuvant group shows the highest IgG titers, followed by AB801 group, and aluminum hydroxide plus CpG 1018 group. Immunization of delta S protein with adjuvant aluminum hydroxide/CpG1018 (50 pg/10 pg) elicited IgG titers in 105 range, whereas with AB801-ISCOM (10 pg) as an adjuvant, the IgG titers were in the range from 106 (against omicron strain) to 107 (against delta strain) (Figure 6B).

[0058] In addition, the effects of adjuvant (AB801 and AB801-ISCOM) on the elicitation of immune responses were shown to be in a dose dependent manner, in which adjuvant at 10 pg elicited higher IgG titers than the adjuvant at 5 pg. When comparing the immunogenicity among treatment groups, the 5 pg AB801-ISCOM adjuvant group showed similar IgG titer to 10 pg AB801 adjuvant group suggesting an approximately two-fold stronger adjuvant activity of AB801-ISCOM compared to AB801 alone, albeit not statistically significant.

[0059] The omicron strain S protein has the highest number of mutations compared to other variants with over 30 mutations, thereby giving the omicron virus the better capability of escaping current vaccines. As a result, vaccines that were developed with different variant S protein, the effectiveness of the vaccines is compromised against the omicron virus (Ou J et al., 2022). This phenomenon is also observed in our study, as the anti-omicron S protein IgG titers were about 106 compared to 107 anti-delta or anti-wild type S protein IgG (Figure 6B).

[0060] Pseudovirus production and pseudovirus-based neutralization assay were performed by RNAi Core Facility of Academia Sinica. Briefly, SARS-CoV-2 pseudoviruses expressing full-length spike protein variants including D614G, B.l.1.7, 501Y.V2, P.l, B.1.617.2, BA.l, BA.2 and BA.4/BA.5 were generated by co-transfecting HEK293T cells with pCMV-AR8.91, pLAS2w.Fluc.Ppuro, and spike protein encoding plasmid. For neutralization assay, HEK293T- hACE2 cells were seeded with density of 1.0x104 cells/well in 96 well white plates and incubated at 37 °C for 16 to 18 hours. Mouse sera were heat inactivated at 56 °C for 30 min followed by 2- fold serial dilution up to a dilution factor of 1:250. The diluted sera were mixed with 1000 transduction unit pseudovirus at equal volumes to give the final sera dilutions at 1:500 followed by incubating at 37 °C for 1 hour. After the incubation, the mixtures were added to the pre- seeded HEK293T-hACE2 cells and incubated at 37 °C for 72 hours. The luminescence emission was triggered by the addition of Bright-Glo-Luciferase reagent (Promega) and the relative luciferase units (RLU) were determined based on the level of luminescence detected by microplate reader (Infinite F500, Tecan). Pseudovirus only and cells only were employed as the controls for 0% and 100% inhibition, respectively. The 50% inhibition dilution titers (ID50) were expressed as 50% neutralization titer (NT50) and calculated (n=3) based on the fold of the serum dilution required to obtain a 50% reduction in RLU compared to control. Geometric mean titers (GMT) were determined by GraphPad Prism version 6 software. One way ANOVA with Tukey's multiple comparison test was used to calculate significance.

[0061] Serum samples harvested on day 28 were also evaluated for neutralization activity against pseudovirus D614G, alpha (B.l.1.7), beta (B.1.351), gamma (P.l), delta (B.1.617.2), and omicron (BA.l and BA.2) strains. Neutralization activity was presented as pNT50 which represents the potency of the induced IgG at specific fold of serum dilution that can inhibit 50% of pseudovirus infection. The pseudovirus neutralization activity is consistent with the level observed in IgG titers, where the AB801-ISCOM adjuvanted group showed the highest neutralization activity compared to AB801 or aluminum hydroxide plus CpG 1018 across all the variants tested (Figure 6C).

[0062] In this study, the neutralization activities of antibodies elicited by OBI-BCVax were tested against different variants. Lower neutralization activities were observed with beta and omicron strains, which is similar to previous publications that vaccines are less effective versus beta or omicron strains (Lefevre B et al., 2021). However, despite the lowered activity, the neutralization activity against omicron BA.l and BA.2 variants was still able to achieve nearly 104 range in the AB801-ISCOM adjuvant group at the dose of 10 pg. This result suggests OBI- BCVax with delta S protein as the antigen and AB801-ISCOM as the adjuvant may be able to provide protection against most of existing variants including BA.l and BA.2 (Figure 6D). The results showed OBI-BCVax induced the strongest neutralization activity against delta strain, followed by alpha, D614, gamma, omicron BA.l, BA.2, and beta strain in the order of potency.

[0063] 4-2: Cytokine analysis and T cell response

[0064] The splenocytes harvested from immunized mice were grinded into a single cell suspension. The suspended cells were seeded on 96-well U-bottom plates at density of 2x10 ^s cells/well and stimulated with SARS-CoV-2 SI mixed peptide pools (2 / g/mL, Mabtech) for 18- 20 hours. The resulting cells were harvested for surface and intracellular markers staining. Cells were first incubated with anti-CD4 and anti-CD8 antibodies for surface staining followed by intracellular staining with anti-IFN-y, anti-IL-4, and anti-Granzyme B antibodies (Biolegend). All flow cytometry data was acquired by Navious EX flow cytometer (Beckman Coulter) and analyzed by Kaluza software.

[0065] Murine IFN-y, IL-2, and IL-4 ELISPOT were performed following the kit instructions (Mabtech). In brief, splenocytes harvested from immunized mice were grinded into a single cell suspension. The suspended cells were seeded in 96-well ELISPOT plates at density of 250,000 cells/well in duplicates. Cells were stimulated with 0.4 pg/well SARS-CoV-2 SI mixed peptide pools (Mabtech) at 37 °C for 18-20 hours before counting the spot numbers by Mabtech ASTOR ELISpot reader.

[0066] In addition to assessing the antibody titers and neutralization activity, we also evaluated the T cell responses after OBI-BCVax immunization. The splenocytes were harvested from immunized mice on day 28 for CD4 and CD8 T cell population analysis. Compared to aluminum hydroxide plus CpG 1018 group, the CD4 ⁺ IFN-y ⁺ population was significantly increased at dose of 10 pg for both AB801 and AB801-ISCOM adjuvant groups. For CD4 ⁺ IL-4 ⁺ population, no differences were noted across the treatment groups. AB801-ISCOM groups were shown to induce CD4 ⁺ IFN-y ⁺ and CD4 ⁺ IL-4 ⁺ cell population in a dose-dependent manner, but not for AB801 groups (Figure 7A).

[0067] For CD8 T cell, compared to aluminum hydroxide plus CpG 1018 group, both AB801 and AB801-ISCOM adjuvant groups (5 and 10 pg) showed significant higher level of CD8 ⁺ IFN-y ⁺ . In addition, AB801-ISCOM adjuvant group (10 pg) showed greater enhancement of CD8 ⁺ IFN-y ⁺ population than AB801 group (10 pg). Furthermore, a marked high level of CD8 ⁺ Granzyme B ⁺ cells was detected in AB801-ISCOM adjuvant group (Figure 7B), whereas no enhancement of CD8 ⁺ Granzyme B ⁺ cells was observed with AB801 group. Taken together, these results suggest that ISCOM plays an important role in triggering T cell responses.

[0068] Given the crucial role of Thl and Th2 in the immune responses, where Thl-type cytokines tend to produce the proinflammatory responses responsible for killing intracellular pathogens and Th2 cells are implicated in the defense against extracellular pathogens. Thl effector cells are characterized by the production of inflammatory cytokines such as IFN-y, TNF- a, and IL-2, capable of stimulating the macrophages, the NK cells, CD8 ⁺ cytotoxic T cells. Th2 effector cells are characterized by the production of anti-inflammatory cytokines such as IL-4, IL- 5, and IL-13. For an effective vaccine, it is important to have high level Thl responses to induce cytotoxic T cells.

[0069] As a result, we next analyzed cytokine production profiles. Splenocytes harvested on day 28 were stimulated with S protein peptide pool and the level of for IL-2 (Thl), IFN-y (Thl), and IL-4 (Th2) secreted from cells were evaluated by ELISpot assay. The result showed that AB801 or AB801-ISCOM adjuvant groups induced IL-2 in a dose dependent manner and the level is higher compared to aluminum hydroxide plus CpG 1018 adjuvant group. Among the treatment groups, at the dose of 10 pg, AB801-ISCOM group showed the highest level of IL-2 induction with around two to eight folds higher than AB801 group and aluminum hydroxide plus CpG 1018 group, respectively. Similar pattern was observed with the IFN-y level, where AB801-ISCOM (10 pg) induced the highest level of IFN-y among the treatment groups (approximately two folds higher than AB801 group), suggesting the potent effects of ISCOM on the induction of Thl responses. In addition, the IL-4 ELISpot results also showed that the spot numbers were higher at the dose of 10 pg for both AB801 and AB801-ISCOM adjuvant groups compared to aluminum hydroxide plus CpG 1018 adjuvant group, yet the extend of enhancement was not as high as IL-2 and IFN-y (Figure 7C). To further assess the polarization of T cell response (Thl or Th2) of the OBI-BCVax candidates, the ratio of I FN-y/l L-4 of ELISpot was determined and shown in Figure 7D. When the ratio is greater than 1, the vaccine is considered to induce greater Thl responses than Th2 responses. In this study, both AB801 and AB801-ISCOM at 10 pg showed the ratio to be 4 and 7, respectively, indicating both types of adjuvants can induce significant higher level of Thl responses.

[0070] 4-3: Immunogenicity study for OBI-BCVax as a booster

[0071] In the immunogenicity study for BCVax as a booster, BALB/c mice were administered delta S protein (10 pg) with adjuvant AB801-ISCOM (2, 5, or 7.5 pg) via intramuscular injection at Day 0 and Day 14. Five out of eight mice were sacrificed on Day 28 and the rest three mice were continue monitored till Day 84 to assess the duration of the immune response. In addition, a group of animals (n=5) that received two prior injections were given a booster with antigen delta S protein (10 pg) and adjuvant AB801-ISCOM (7.5pg) on Day 56 and sacrificed 4 weeks later on Day 84. Treatment details are shown in Figure 8A. For immunogenicity analysis, blood samples were collected from all animals before each dosing and on Days 14, 28, 56, 70, and 84. Spleens were collected from animals sacrificed on Day 84. [0072] Since majority of global population have taken at least two doses of COVID-19 vaccination, BCVax was also evaluated for the potential as a booster. In addition to the groups of mice receiving two injections on day 0 and day 14, a group of mice with a third injection on day 56 was included in the study. The dosing scheme is shown in Figure 8A. Five out eight mice which received two vaccinations were sacrificed at day 28 and the rest of three mice were maintained for continue monitoring the IgG titer and neutralization activity to assess the durability of immune responses after two doses of vaccination. Serum samples from each group were harvested on the schedule shown in Figure 8A for evaluation of anti-S protein IgG titer and neutralization activity against existing variants. The group with booster showed elevated IgG titers against delta and BA.2 strains, more importantly the IgG titer against BA.5 strain was significantly enhanced on day 84 compared to mice only received two injections (Figure 8B). The time course of the IgG titer was shown in Figure 8C. For the mice receiving two injections, IgG titers reached peak level at week 4 and sustained at the same level until sacrifice at week 12 (Figure 8C). In the mice receiving a booster, the IgG titers were further stimulated to higher levels, not only to delta and BA.2 strains, but also to BA.5 strain (Figure 8B and 8C).

[0073] The pseudovirus neutralization activity of booster was also evaluated against delta (B.1.617.2) and omicron (BA.2 and BA.4/BA.5) strains using serum samples collected on Day 84. Consistent with the findings on IgG titers, the booster elicited higher neutralization activity than the group with only two injections as shown in Figure 8D. The neutralization activity was not statistically different against delta strain either with or without booster. However, although the neutralization activity against BA.2 or BA.4/BA.5 strains was limited in mice only received two injections, the neutralization activity reached 10 ⁴ level for the mice received the booster (Figure 8D).

[0074] Serially diluted sera were incubated with SARS-CoV-2 Delta variant (Taiwan Centers of Disease Control, strain No.1144) or Omicron BA.l (Taiwan Centers of Disease Control, strain No.16804) at 37 °C for 1 hour. The number of infectious virus particles were used in 100 folds of Median Tissue Culture Infectious Dose (TCID50). The mixtures were then added to pre-seeded Vero E6 cells for 4 days incubation. Cells were fixed with 10% formaldehyde and stained with 0.5% crystal violate for 20 minutes. The plates were washed with tap water and scored for infection. The 50% protective titer was calculated by Reed and Muench Method. The live virus neutralization titers of sera collected at day 84 of immunized mice against SARS-CoV-2 Delta and BA.l variants is as illustrated in Fig. 8E. Similar trend was observed as pseudovirus neutralization activity tests, suggesting BCVax induced immunity is capable to neutralize both Delta and Omicron strains infection.

[0075] For the T cell population analysis, the CD8 ⁺ IFN-y ⁺ and CD8 ⁺ Granzyme B ⁺ populations were also enhanced after booster received (Figure 8F). Taken together, these results suggest BCVax has a promising potential as a booster with a broad coverage against various variants.

[0076] While specific aspects of the invention have been described and illustrated, such aspects should be considered illustrative of the invention only and not as limiting the invention as construed in accordance with the accompanying claims. All publications and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

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