TWI843471B - Composition comprising antigen and dna and use thereof - Google Patents

Abstract

A composition is provided, wherein the composition includes a subunit vaccine including a first amount of a subunit, and a nucleic acid vaccine including a second amount of a vector.

Description

Composition containing antigen and DNA and its use

The present disclosure provides a composition and its use, in particular a composition containing an antigen and DNA and its use.

Many vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been tested in clinical trials and some have been used for mass vaccination; however, the development of safe, effective and affordable vaccines is still an ongoing process. Based on the results of the development of COVID-19 vaccines across multiple platforms, the spike glycoprotein on the surface of the virus has been confirmed to be an ideal immunogen to induce a protective immune response.

Among all vaccine platforms, DNA and mRNA vaccines have unique advantages, including rapid design and production, cost-effectiveness, and easy manipulation of coding sequences to target emerging mutations. Importantly, their mechanism of action enables the introduction of target antigens into the host immune system in a manner that mimics natural infection. More importantly, in vivo production of the desired antigen by antigen-presenting cells (APCs) can promote intracellular antigen processing and presentation by class I major histocompatibility complex (MHC)-activated CD8+ T cells, typically leading to a CD4+ type 1 T helper 1 cell (Th1)-mediated immune response and avoiding the Th2-biased immune response associated with complications of vaccine-associated enhanced respiratory disease (VAERD). CD8+ T cells also play an important role in inducing cross-protective immune responses against SARS-CoV-2 variants. In addition, DNA vaccines have more advantages than mRNA vaccines; DNA vaccines do not require additional in vitro transcription steps or ultra-low temperature storage, which limits the global transportation of mRNA vaccines.

However, the obstacles to DNA vaccines are the difficulty in effectively entering cells and the limited amount of antigen production, which leads to low immunogenicity and poor antigen-specific antibody response. To ensure the efficacy of DNA vaccines, electric pulses or gene guns are usually used to promote the delivery of plasmid DNA to the nucleus of host cells for the expression of encoded proteins.

In contrast, subunit protein vaccines containing specific viral proteins are more effective in directly stimulating antigen-specific B cell-mediated antibody secretion than in inducing T cell-mediated immune responses, which require further protein-to-peptide processing. Typically, potent adjuvants must be incorporated into subunit vaccines to carry antigens to APCs or stimulate helper T cell function.

Since DNA and protein vaccines utilize different mechanisms to elicit an immune response, combining the advantages of these two vaccines may overcome the disadvantages of each vaccine type. Therefore, there is an urgent need to provide a composition that mitigates and/or avoids existing disadvantages.

The present invention provides a composition, including: a subunit vaccine, including a first dose of a subunit; and a nucleic acid vaccine, including a second dose of a vector.

In one aspect of the present invention, the subunit of the subunit vaccine may include pathogen components, recombinant proteins, polysaccharides, peptides or a combination thereof.

In one aspect of the present invention, the subunit of the subunit vaccine may include the recombinant protein.

In one aspect of the present invention, the subunit of the subunit vaccine may include recombinant SARS-CoV-2 protein.

In one aspect of the present invention, the subunit of the subunit vaccine may include recombinant SARS-CoV-2 trimeric spike (rTS) protein.

In one aspect of the present invention, the recombinant SARS-CoV-2 trimeric spike (rTS) protein may include a trimerization domain of IZN4.

In one aspect of the present invention, the vector included in the nucleic acid vaccine may be a plasmid encoding a protein sequence that is the same as or different from the recombinant protein included in the subunit vaccine.

In one aspect of the present invention, the vector can be a plasmid encoding the SARS-CoV-2 trimerized spike (TS) sequence.

In one aspect of the present invention, the subunit vaccine may further include a third dose of an adjuvant.

In one aspect of the present invention, the adjuvant may be an aluminum salt. Preferably, the adjuvant is aluminum hydroxide (Al(OH) ₃ ), aluminum phosphate (AlPO ₄ ) or aluminum hydroxy oxide (AlO(OH)). More preferably, the adjuvant is aluminum hydroxide (Al(OH) ₃ ).

In one aspect of the present invention, the first dose may be in the range of 0.1 μg to 10 μg, preferably in the range of 0.1 μg to 5 μg, and most preferably in the range of 0.1 μg to 1 μg.

In one aspect of the present invention, the second dose may be in the range of 1 μg to 300 μg, preferably in the range of 1 μg to 200 μg, and most preferably in the range of 5 μg to 100 μg.

In one aspect of the present invention, in the composition of claim 8, the third dose may be in the range of 100 μg to 500 μg, preferably in the range of 150 μg to 400 μg, and most preferably in the range of 200 μg to 300 μg.

In one aspect of the present invention, the adjuvant in the composition of claim 1 is an aluminum salt.

The present invention further provides a method for preventing or ameliorating a disease in a subject in need thereof, comprising administering to the subject a preventive effective amount of the above composition.

In one aspect of the present invention, the disease may be caused by a virus.

In one aspect of the present invention, the virus may belong to Retroviridae , Parvoviridae , Paramyxoviridae , Coronaviridae or Herpesviridae . Preferably, the virus belongs to Coronaviridae.

In one aspect of the present invention, the virus may be severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) or severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Preferably, the virus is severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2).

In one aspect of the present invention, the preventively effective amount may be in the range of 0.1 mg to 10 mg, preferably in the range of 0.1 mg to 5 mg, and most preferably in the range of 0.1 mg to 2 mg for the subject in need.

In one aspect of the present invention, the composition may further include stabilizers, suspending agents, preservatives, surfactants, dissolution adjuvants, pH adjusters or aggregation inhibitors.

In one aspect of the present invention, the subject can be an animal subject, preferably a mammalian subject, and more preferably a human subject.

The present invention further provides the use of the above composition in the preparation of drugs for preventing diseases.

The present invention further provides a method for treating a disease in a subject in need thereof, comprising administering a preventive effective amount of the above composition to the subject.

The present invention further provides a method for enhancing the humoral response of a subject in need thereof, comprising administering a preventive effective amount of the above composition to the subject.

The present invention further provides a method for inducing a Th1-dominant immune response in a subject in need thereof, comprising administering to the subject a preventive effective amount of the above composition.

The present invention further provides a method for improving S-specific T cell response in a subject in need thereof, comprising administering to the subject a preventively effective amount of the above composition.

The present invention further provides a method for enhancing antibody response and protective effect in a subject in need thereof, comprising administering to the subject a preventively effective amount of the above composition.

The present invention further provides a method for enhancing antigen-specific T cell response in a subject in need thereof, comprising administering to the subject a preventively effective amount of the above composition.

In one aspect of the present invention, the antigen can be ovalbumin (OVA) or trimeric spike protein.

The following will be described in detail with reference to the drawings to make other novel features of the present disclosure more apparent.

Different embodiments of the present invention are provided in the following description. These embodiments are used to explain the technical content of the present invention and are not intended to limit the scope of the present invention. The features described in one embodiment can be applied to other embodiments through appropriate modification, replacement, combination or separation.

As used herein, the terms "a," "an," and "one" mean "at least one," unless otherwise specified.

Unless otherwise specified, the terms "cytotoxic T lymphocytes", "cytotoxic T cells" and "CTL" are used interchangeably in the present invention.

As used herein, the terms "prevent," "prevention," or "prophylaxis" include any measure that reduces mortality or morbidity. Prevention can be classified as primary, secondary, or tertiary. Primary prevention is aimed at avoiding the occurrence of disease, and secondary and tertiary prevention include measures to prevent the progression of disease and the onset of symptoms, restore function and alleviate disease-related symptoms, thereby reducing the adverse effects of existing disease. Alternatively, prevention can include a wide range of preventive treatments aimed at reducing the severity of a specific condition, for example, reducing clinical symptoms such as fever, wheeze, cough, and upper respiratory tract infections.

The composition of the present invention can also be used as a vaccine. In the present invention, the term "vaccine" (also referred to as "immunogenic composition") refers to a composition that has the function of inducing an immune response when inoculated into an animal.

Any composition described herein can be formulated to be suitable for various routes of administration, such as intravenous, intraarticular, intraconjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal or subcutaneous administration. The composition can be an aqueous solution or a lyophilized formulation.

Materials and methods

Production of rTS

To produce a secreted SARS-CoV-2 S protein (rTS protein) in a prefusion form, the DNA fragment encoding the Wuhan strain S protein was designed to contain a non-functional furin cleavage site (R682G, R683S, R685S) and two stabilizing prolines in the hinge loop (K986P, V987P). In addition, the transmembrane domain and the C-terminal intracellular tail were removed (S-2P) or replaced by the trimerization domain IZN4 (S-Trimer), and then purified with histidine (8-mer) at the carboxyl terminus. Both S variant DNA constructs were codon-optimized for modern Homo sapiens, synthesized, and cloned into the pcDNA3.1(+) plasmid vector by GenScript (Piscataway, NJ, USA). Production of the S-trimer was compiled using the ExpiCHO™ Expression System kit (ThermoFisher Scientific, Carlsbad, CA, USA). Briefly, the S-trimer (or S-2P) was transiently expressed by ExpiCHO cells in serum-free medium according to the manufacturer's instructions. The culture medium containing S-trimer was centrifuged at 15,000 rpm for 30 minutes at 4 ^° C; subsequently, the supernatant was dialyzed into equilibration buffer (50 mM Tris-HCl, 150 mM NaCl, and 20 mM imidazole; pH 8.9). S-trimer was purified by equilibrated Ni ²⁺ -NTA agarose column (GE). Finally, S-trimer was eluted by equilibration buffer containing 0.5 M imidazole and dialyzed against imidazole-free buffer (20 mM sodium phosphate; pH = 8.0).

Production and characterization of DNA vaccine plasmids

For the construction of pVax-TS, the sequence of TS was human codon usage optimized, synthesized by Genscript (Piscataway, NJ, USA) and cloned into the DNA vaccine vector pVax1 (Thermo Fisher, Waltham, MA, USA) at the NheI-NotI sites (Figure 6(E)). Plasmid DNA for animal vaccination was amplified in competent DH5α cells and purified using an endotoxin-free plasmid extraction kit (Qiagen, Redwood, CA, USA). Final plasmid stocks were verified by restriction enzyme digestion followed by DNA fragment size determination by agarose gel electrophoresis. The expression of rTS from pVax-TS was confirmed by transfecting 293 T cells and then comparing with the empty vector pVax1 by immunoblotting using anti-S antibody (Mab5) (Figure 7).

Animal vaccination

BALB/c and C57BL/6 mice and Syrian hamsters were obtained from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). Mice or hamsters aged 6 to 12 weeks were used. Anesthetized animals were injected intramuscularly every 3 weeks with various vaccines, including DNA alone, protein in aluminum hydroxide (aluminum), or a combination of DNA and protein in aluminum. Blood samples were collected from mice and hamsters by tail vein or submandibular bleeding. All animals were housed at the Animal Center of the National Health Research Institutes (NHRI) and cared for in accordance with the institutional animal care protocol. All experimental animal protocols were approved by the NHRI Institutional Animal Care and Use Committee (IACUC) (protocol number: NHRI-IACUC-109077-A).

Neutralizing Antibody Analysis

Neutralizing antibody titers against live SARS-CoV-2 were assessed by TCID ₅₀ assay. Live virus (hCoV-19/Taiwan/4/2020) was generated as follows. SARS-CoV-2 variants (hCoV-19/Taiwan/4/2020) were obtained from the Taiwan Centers for Disease Control (CDC). Viruses were amplified in Vero cells grown in M199 medium supplemented with 2 μg/mL TPCK-trypsin (Sigma) at 37 ^° C. Neutralizing titers were determined as the reciprocal of the serum dilution required to prevent 50% infection in quadruplicate inoculations. For calculation purposes, neutralization titers below a 1:20 starting dilution were assigned a value of 10.

Cytokine assay

Cytokine production by splenocytes was assessed using a cytokine sandwich ELISA. Mouse spleens were harvested 7 days after the second vaccination. Splenocytes were collected by filtration through a 70 μm cell strainer after mechanical homogenization through a mesh into RPMI-1640 medium and suspension in LCM containing 10% fetal bovine serum (FBS). The splenocytes obtained were stimulated with recombinant SARS-CoV _- 2 S (10 μg/ml) in 24-well plates (5 × 10 ⁶ cells/well) at 37 ^o C and 5% CO 2. After 3 days, supernatants were collected to quantify the amount of Th1 (IFN-γ and IL-2) and Th2 cytokines (IL-13, IL-5, and IL-4) produced by using different cytokine ELISA kits (Invitrogen, Waltham, MA, USA) as described in the manufacturer's instructions.

ELISPOT test

To characterize SARS-CoV-2 spike-specific T cell responses, spleens of mice were harvested one week after the second vaccination. The number of IFN-γ or IL-2 secreting cells in the spleen of each mouse was assessed using a mouse IFN-γ ELISPOT assay kit (BD Biosciences) according to the manufacturer's instructions. Splenocytes (5 × 10 ⁵ cells/well) were inoculated in duplicate into wells containing 10 μg/ml recombinant S-2P or synthetic peptides (S _444-458 KVGGNYNYLYRLFRK (SEQ ID 1) and S _535-543 KNKCVNFNF (SEQ ID 2)), which have been reported as CD4 ⁺ and CD8 ⁺ T cell epitopes in BALB/c mice. After restimulation of spleen cells for 2 days at 37 ^° C and 5% CO ₂ , the cells were removed from the plates by washing three times with PBST. Then, the plates were incubated with biotinylated detection antibodies (1:250) at 37 ^° C for 2 hours. Each well was filled with staining solution (3-amine-9-ethylcarbazole, Sigma-Aldrich, Burlington, MA, USA) to visualize any spots. After 30 minutes, the plate was placed under tap water to stop the reaction. The spots were counted using an ELISPOT analyzer (Cellular Technology Ltd., Shaker Heights, OH, USA).

Intracellular cytokine staining

Splenocytes were collected 7 days after the second vaccination. Single cell suspensions (1 × 10 ⁶ cells/200 μl) were cultured in a round-bottom 96-well microplate in the presence of a SARS-CoV-2 S protein peptide pool for 24 hours (Table 1). Cell surface markers were stained for 30 minutes at 4 ^o C using the following anti-mouse monoclonal antibodies: anti-CD3e-PerCP/Cy5.5 (BioLegend, clone: 145-2C11), anti-CD4-FITC (BD Biosciences, clone: RM4-5), anti-CD8-BV650 (BioLegend, clone: 53-6.7), and anti-CD19-BV510 (BioLegend, clone: 6D5). Then, cells were fixed and permeabilized using Lysis Solution and Permeabilizing Solution (BD Biosciences) according to the manufacturer's instructions, followed by staining with anti-IFN-γ-PE (BD Biosciences, clone: RM4-5) and anti-TNF-α-APC (eBioscience, clone: MP6-XT22) for 1 hour at 4 ^° C. All samples were acquired using an Attune NxT flow cytometer (Thermo Fisher Scientific) and analyzed using FlowJo software v10.6.0.

Table 1. SARS-CoV-2 spike-specific T cell epitopes in BALB/c mice Name Peptide sequence CD4 T cell epitope S61-75 NVTWFHAIHVSGTNG (SEQ ID 3) S339-363 AWNRKRISNCVA (SEQ ID 4) S444-458 KVGGNYNYLYRLFRK (SEQ ID 5) CD8 T cell epitopes S268-275 GYLQPRTF (SEQ ID 6) S535-543 KNKCVNFNF (SEQ ID 7) S1052-1060 FPQSAPHGV (SEQ ID 8)

Viral challenge of Syrian hamsters

Animal models of SARS-CoV-2 challenge were performed in Syrian hamsters as described previously. Briefly, Syrian hamsters were immunized IM with PBS (negative control), 100 µg pVax-ST, 0.5 µg ST/Al(OH) ₃ , or ST/Al(OH) ₃ mixed with pVax-ST. The injection volume per PBS control and combination vaccine was 100 μl per IM dose. Hamsters were challenged intranasally with 1 x 10 ⁵ TCID ₅₀ dose of SARS-CoV-2 on day 45. Four hamsters per group were sacrificed on day 3 after SARS-CoV-2 virus challenge for viral load quantification. The body weight of the other four hamsters in each group was recorded daily until sacrifice on day 6. To determine the viral load in the lungs, left lung tissue was homogenized in 2 mL PBS using a gentle-MACS® dissociator (Miltenyi Biotec). After centrifugation at 600 x g for 5 min, the clear supernatant was collected for live virus titration (TCID ₅₀ assay) and viral RNA quantification.

Hematoxylin and eosin (H&E) staining

To evaluate lung tissue pathology by H&E staining, lung tissues from hamsters were fixed in 10% formalin and embedded in paraffin. Sections were prepared and stained with H&E. Lung pathology, including the overall extent of lesions, lung cell hyperplasia, and inflammatory infiltration, was evaluated by clinical pathologists at the core pathology facility of NHRI (Miaoli, Taiwan). The score was determined based on the percentage of inflamed area in each section of the total lung lobe collected from each animal in each group by using the following scoring system: 0 = no pathological changes; 1 = infiltration area ≤ 10%; 2 = infiltration area 10%; 3 = infiltration area ≥ 50%. When pulmonary edema and/or alveolar hemorrhage were observed, an additional point was added. The total score for all lobes in the image is shown for individual animals.

Statistical analysis

Statistical data were analyzed using GraphPad Prism software. Two-tailed Mann-Whitney test was used to compare two experimental groups. Multiple groups were compared using Kruskal-Wallis ANOVA and Dunn's multiple comparisons test. Two-way ANOVA was applied to multiple groups at different time points. P values < 0.05 were considered significant. ns, not significant.

Embodiment 1

rTS protein and DNA containing alum preparations enhanced humoral responses in mice.

An rTS protein was designed that maintains a stable trimer structure with a 2P modification and a C-terminus fused to the trimerization domain IZN4. To test whether the immunogenicity of the rTS/mineral subunit vaccine could be improved by mixing with a plasmid encoding the TS sequence, a TS expression plasmid pVax-TS was constructed by inserting the TS coding sequence into pVax1, a vector commonly used in DNA vaccine development (Figure 6).

First, different doses (5 μg, 20 μg, or 100 μg) of pVax-TS and rTS/Alum were prepared as described in Materials and Methods. BALB/c mice (n=5-6/group) were immunized twice IM at 3-week intervals; serum samples were collected and analyzed by enzyme immunosorbent assay (ELISA) to determine the anti-S total IgG antibody titer (Figure 1(A)). The results showed that immunization with rTS/Alum containing 5 μg or 20 μg of pVax-TS induced the production of anti-S IgG like the rTS/Alum group, while immunization with the formulation containing 100 μg of pVax-TS at the indicated time points increased the anti-S IgG titer to a level significantly higher than that of the rTS/Alum group (Figure 1(B)). In addition, to evaluate the contribution of 100 μg of pVax-TS plasmid DNA to the immunity induced by this combination vaccine, BALB/c mice were immunized with pVax-TS alone or with pVax-TS/alum or pVax-TS/rTS combinations. IM immunization with 100 μg of pVax-TS induced higher anti-S IgG titers than PBS control immunization (40x, 70x, and 5x on days 28, 42, and 56, respectively). However, immunization with pVax-TS mixed with alum adjuvant or rTS did not further enhance anti-S IgG titers, which increased significantly only in mice receiving the pVax-TS + rTS/alum combination vaccine and peaked three weeks after the second vaccination (day 42) (Figure 1(C)). This result indicates a synergistic enhancement of the anti-S IgG response induced by conjugating rTS/alum to pVax-TS DNA.

To investigate the humoral responses induced by the TS DNA+protein combination vaccine, sera collected on day 42 were analyzed for total anti-S IgG (Figure 2(A)), anti-S IgGl (Figure 2(B)), anti-S IgG2a (Figure 2(C)), and SARS-CoV-2 neutralization (NT) titers (Figure 2(E)). In addition, the combination of empty pVax1 and rTS/alum was included for comparison to evaluate the adjuvant effect of bacterioplasmic DNA. Although intramuscular (IM) injection of pVax-TS (100 μg) induced relatively low anti-S total IgG titers and IgG2a-biased humoral responses with a wide range of IgG2a/IgG1 ratios (Figure 2(D)), it also elicited NT titers equivalent to those induced by rTS/alum. Importantly, vaccination with pVax-TS+rTS/alum induced the highest titers of S-specific total IgG, IgG1, and IgG2a, the highest IgG2a/IgG1 ratio, and significantly higher NT titers compared to other vaccination regimens. However, pVax-only formulated with rTS/alum did not enhance antibody responses in mice, but did induce significantly higher IgG2a titers and higher IgG2a/IgG1 ratios compared to rTS/alum, suggesting that Th1-biased immune responses were present despite the fact that alum generally induces Th2-biased immunity. These data may be attributed to the fact that pVax-TS exhibits a Th1-directed adjuvant effect through its backbone structure, while the expression of additional TS antigens adjuvanted with alum at the injection site can enhance S-specific antibody responses.

Embodiment 2

The rTS/alum vaccination formulated with TS DNA induced a Th1 dominant immune response.

To further evaluate the cytokine profile of S-specific T cells in the spleen, BALB/c mice (n=6/group) were immunized with the different vaccine regimens described above. One week after the second vaccination, changes in the amount of IFN-γ, IL-2, IL-4, IL-5, and IL-13 (Figures 3(A)-3(E)) in the supernatant of stimulated spleen cells were measured by ELISA. As expected, due to the limited amount of Th1 cytokines (IFN-γ and IL-2), the alum adjuvant rTS produced a Th2-biased cytokine profile with a relatively low Th1/Th2 cytokine ratio, while a greater amount of Th2 cytokines (IL-4, IL-5, and IL-13) secretion was detected in the spleen cells of rTS/alum-vaccinated mice. Compared with the rTS/alum group, the pVax-TS alone group and the rTS/alum plus pVax group did not show a significant increase in the amount of Th1 cytokines, but showed partial inhibition of Th2 cytokine secretion, resulting in a Th1-biased response with a higher Th1/Th2 cytokine ratio (Figures 3(F)-3(H)). The IFN-r and IL-2 secretions of spleen cells in the pVax-TS+rTS/alum vaccination group were the highest among all groups, the IL-4 and IL-13 cytokine secretions were comparable to those in the rTS/alum group, and the Th1/Th2 cytokine ratio was relatively high (Figures 3(F)-3(H)). These findings suggest that the pVax-TS+rTS/alum regimen substantially induces a mixed Th1/Th2 immune response, in which the Th1 immune response is dominant.

Embodiment 3

Vaccination with rTS/alum plus pVax-TS improves S-specific T cell responses.

Since Th1-mediated immune responses are often associated with the induction of cellular immunity, the development of Spike-specific T cells in mice after immunization with TS DNA+protein combination vaccines was investigated. BALB/c mice (n=8/group) were immunized IM at 3-week intervals and sacrificed one week after the second vaccination. Splenocytes isolated from each group of mice were then restimulated ex vivo with the Spike extracellular domain, CD4+ T cell epitope (S444-458), or CD8+ T cell epitope (S535-543). S-specific cell responses of splenocytes were assessed by ELISpot to quantify the number of IFN-γ-secreting cells (Figure 4(A)).

After two days of culture in complete medium, the mean frequency of IFN-γ-secreting cells was less than 20 cells/106 in all groups, including the PBS control group stimulated with S protein or peptide. Consistent with the cytokine production profile, immunization with the rTS/alum regimen produced the lowest number of S- and S444-458-specific IFN-γ-secreting T cells in the spleen (58.25±43.34 and 32.25±8.45) and the least responsive CD8+ T cells to epitope stimulation (21.75±8.8).

Immunization with pVax-TS DNA alone produced significantly increased amounts of S- and S535-543-specific IFN-γ-secreting cells (149.1±96.52 and 114.1±81.64), with significant inter-individual variability. In addition, BALB/c mice that received the pVax-TS+rTS/alum combination vaccine produced significantly higher frequencies of S- and S444-458-specific T cells than other mice (183.6±75.08; 123.9±37.98); when stimulated with a CD8+ T cell epitope (S535-543), their spleen cells had a number of IFN-γ-secreting cells (66.75±45.7) comparable to the pVax-TS-only group. Notably, intramuscular (IM) vaccination with pVax-TS DNA induced cellular immunity that was biased toward CD8+ T cell responses. In addition, the combination of alum-adjuvanted rTS with pVax-TS induced a more robust CD4+ T cell response without significantly impairing the development of CD8+ T cell immune responses.

Next, the ratio of effector CD4+ (Figure 4(B)) and CD8+ (Figure 4(C)) T cells induced by different vaccination schedules was verified. After 24 hours of restimulation with a pool of S-specific T cell epitopes (Table 1), spleen cells were stained with antibodies targeting T cell markers and cytokines by intracellular staining (ICS) (Figure 8). Flow cytometric analysis showed no statistically significant differences between the groups receiving pVax-TS alone or rTS/alum one week after the second vaccination. However, mice vaccinated with pVax-TS+rTS/alum showed a significant increase in the amount of S-specific IFN-γ- and TNF-α-producing CD8+ T cells (Figure 4(C)) and TNF-α+CD4+ T cells. However, the percentage of IFN-γ+CD4+ T cells in the spleen of these mice was similar to that of mice receiving the pVax-TS DNA vaccine (Figure 4(B)). Overall, the combination of pVax-TS and rTS/alum regimens synergistically elicited S-specific CD4+ and CD8+ T cell-mediated immunity.

Embodiment 4

pVax-TS+rTS/alum combination vaccine enhances antibody response and protection in Syrian hamsters.

Based on the mouse data, the DNA+protein combination vaccine regimen was tested in SARS-CoV-2 virus challenge studies. Due to the limited binding affinity of the mouse ACE2 receptor for the SARS-CoV-2 spike protein, the Syrian hamster was used as an alternative SARS-CoV-2 infection model, as the ACE2 receptor of this species binds tightly to the SARS-CoV-2 spike protein and mediates viral entry. In this animal study, hamsters (n=8/group) received two intramuscular injections of PBS control, pVax-TS, rTS/alum, or pVax-TS+rTS/alum, 3 weeks apart, and were infected intranasally with SARS-CoV-2 on day 45 (Figure 5(A)). Serum samples were collected at the indicated time points and antibody responses were evaluated by measuring anti-S IgG titers (Figure 5(B)) and live virus NT titers (Figure 5(C)). Hamsters vaccinated with pVax-TS + rTS/alum vaccine produced significantly increased anti-S IgG and neutralizing antibody titers on days 14, 28, and 42 after the first and booster doses, but no significant differences were detected between hamsters vaccinated with pVax-TS DNA or rTS/alum alone.

In the challenge study, hamsters from four groups were infected with 200 median tissue culture infectious doses (TCID50) of SARS-CoV-2. To analyze lung histopathology and viral load, half of the hamsters in each group (n=4/group) were sacrificed on day 3 post infection; the other half was monitored daily for weight change (%) and then sacrificed on day 6 post infection (dpi). After infection, hamsters vaccinated with rTS/alum alone or with pVax-TS did not lose weight during the first 6 days post infection, while hamsters in the PBS control group gradually lost about 15% of their weight within 6 days post infection. Hamsters vaccinated with pVax-TS DNA alone had a slight weight loss, but the weight loss was significantly less than that of the PBS control group (Figure 5(D)). On day 3 post-infection, viral titers (log 9.61 TCID50/ml) indicated acute viral replication in the lungs of hamsters in the PBS control group. There was no significant reduction in lung viral titers in hamsters that received pVax-TS (log 7.15 TCID50/ml) or rTS/alum (log 6.11 TCID50/ml) alone compared to the PBS control group. However, in the pVax-TS+rTS/alum group, viral replication in the lungs was well controlled below the detectable limit (Figure 5(E)). Histopathological analysis on day 6 post-infection showed that greater inflammation and tissue damage were observed in the lungs of the PBS group. All groups of vaccinated hamsters showed less lung damage than the PBS group, except for the group containing hamsters vaccinated with pVax-TS+rTS/alum, which was not significantly different (Figure 5(F), 5(G)). These findings indicate that protective efficacy is positively correlated with anti-S IgG titers elicited by the vaccine regimen. In summary, these results support the data from the mouse studies and demonstrate that the combination vaccine regimen can elicit a robust humoral immune response to inhibit viral replication and protect hamsters from lung damage.

Embodiment 5

T cell responses were assessed using ovalbumin (OVA).

In the present invention, TS DNA and protein were co-delivered with alum by direct intramuscular injection to improve the immunogenicity of TS, which has been shown to enhance the kinetics and amplitude of anti-S antibody responses, virus neutralization efficacy, and spike-specific T cell responses. To further explore whether the strategy of combining DNA and protein with alum can generally enhance antigen-specific T cell responses, ovalbumin (OVA) was used as an antigen to evaluate the T cell responses of C57BL/6 mice vaccinated twice intramuscularly with pVax-OVA alone, rOVA/alum, or a combination of pVax-OVA and rOVA/alum. Through this experiment, it was confirmed that the DNA+protein combination vaccine enhanced CD4+ and CD8+ T cell immunity regardless of whether the antigen was TS or OVA (Figures 9(A) and (B)). Importantly, conjugation of OVA DNA and protein to alum was found to simultaneously increase the number and effector function of antigen-specific CD8+ T cells (Figure 9(C) and (D)), which was associated with enhanced cytotoxic T-lymphocyte (CTL) killing capacity (Figure 9(E) and (F)).

In the present invention, the immunogenicity and efficacy of alum formulated with recombinant SARS-CoV-2 trimeric spike (rTS) protein and its encoding plasmid were comprehensively evaluated in rodents receiving naked DNA, protein/alum or DNA+protein/alum combination vaccines by direct intramuscular (IM) injection. The results showed that the combination vaccine regimen achieved co-delivery of the two vaccines and synergistically enhanced humoral and cellular responses, which is expected to prevent SARS-CoV-2 infection.

Based on these interim results from a well-designed clinical trial, this simple formulation of rTS protein and plasmid DNA mixed with alum should be safe and easily applicable to induce protective immunity in human clinical trials. In addition, the rapid manufacturing process of DNA vaccines is applicable to a new generation of vaccines using DNA-encoded new variant S combined with the original rTS, which can provide a rapid response to outbreaks of new variants.

The specific embodiments described above should be understood to be exemplary only and not limiting of the remainder of the invention in any way, and features of different embodiments may be mixed and matched as long as they do not conflict with each other.

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FIG1 is a set of graphs showing the synergistic increase in anti-S IgG induced by the combination of TS protein + DNA and alum. (A) A schematic diagram showing the vaccination and serum collection schedule. BALB/c mice (5-6/group) were immunized twice intramuscularly (IM) on days 0 and 21 with S-Trimer/Al(OH) ₃ in the indicated vaccine combination. The PBS injection group was used as a blank control. Serum was collected on days 0, 28, 42, and 56. (B, C) Anti-S IgG titers in serum samples collected at the indicated time points were detected using enzyme immunoassay (ELISA). Each symbol represents the endpoint titer of one mouse; each bar represents the geometric mean ± 95% confidence interval (CI) of the IgG endpoint titer of the group. Statistically significant differences between adjuvant vaccine groups were determined by two-tailed Mann-Whitney test. *P < 0.05, **P < 0.008, ns: not significant. FIG. 2 is a set of graphs showing humoral responses induced by Trimer-S protein + DNA formulated with alum. BALB/c mice (9/group) were immunized twice IM on days 0 and 21 with 100 μg pVax-TS (green symbols), TS/alum (blue symbols), TS/alum combined with 100 μg pVax-1 (yellow symbols), or TS/alum combined with 100 μg pVax-TS (red symbols). PBS was injected as a blank control. (A) Total anti-S IgG titers, (B) anti-S IgG1 titers, and (C) anti-S IgG 2a titers in serum samples (day 42) were measured by ELISA. (D) IgG2a/IgG1 ratios were calculated based on S-specific antibody titers measured by ELISA. Neutralizing activity against live SARS-CoV-2 was assessed in serum samples collected 3 weeks after the second immunization (day 42). Each symbol represents the IgG endpoint titer or the reciprocal 50% inhibition dilution ID50 titer of one mouse; each bar represents the geometric mean ± 95% confidence interval (CI) of the IgG endpoint titer for that group. Statistically significant differences between adjuvant vaccine groups were determined by a two-tailed Mann-Whitney test. *P < 0.05, **P < 0.008, ***P < 0.0005, ****P < 0.0001, ns: not significant. Figure 3 is a set of graphs showing that immunization with TS/alum combined with pVax-TS increases Th1 cytokine production. BALB/c mice (n=6/group) were vaccinated twice with the indicated vaccine combinations at 3-week intervals (day 0 and day 21). Splenocytes were collected one week after the second vaccination (day 28) and cultured with the SARS-CoV-2 S extracellular domain to stimulate cytokine secretion. The amount of secreted Th1 cytokines including (A) IFN-γ and (B) IL-2 and the amount of secreted Th2 cytokines including (C) IL-4, (D) IL-5, and (E) IL-13 were measured by sandwich ELISA. The Th1/Th2 ratio was calculated by dividing the amount of IFN-γ produced by the amount of each Th2 cytokine produced, i.e., (F) IFN-γ/IL-4, (G) IFN-γ/IL-5, or (H) IFN-γ/IL-13. Each symbol represents the cytokine value of one mouse; the cytokine values for each group are expressed as the geometric mean ± 95% confidence interval (CI). Statistical analysis for all comparisons was performed using the two-tailed Mann-Whitney test. *P < 0.05, **P < 0.008, ns: not significant. Figure 4 is a set of graphs showing that TS/alum vaccine formulated with pVax-TS can enhance S-specific T cell responses. Spleens were collected from PBS control-treated or immunized BALB/c mice (n=8/group) one week after the second vaccination. (A) Splenocytes from each individual were cultured in culture medium as a control or stimulated with SARS-CoV-2 spike extracellular domain, CD4+ T cell epitopes, or CD8+ T cell epitopes for two days. The number of IFN-γ secreting cells was assessed by ELISpot. Splenocytes were collected as described above and cultured with S-specific T cell epitopes for 24 hours. Splenocytes were gated on CD3a+/CD19- cells to determine T cell populations using flow cytometry. (B) Percentage of CD4+ T cells producing IFN-γ and TNF-α. (C) Percentage of CD8+ T cells producing IFN-γ and TNF-α. Each symbol represents one mouse; each bar represents the mean ± SD of each group. Statistically significant differences were determined by a two-tailed Mann-Whitney test. * p < 0.05, ** p < 0.008, and *** p < 0.0005 were considered significant; ns: not significant. Figure 5 is a set of graphs showing the immune response and protective efficacy of TS/alum combined with pVax-TS in hamsters. (A) Schematic diagram showing the vaccination, serum sample collection, and SARS-CoV-2 challenge schedule for the hamster study. Hamsters (n=8/group) were vaccinated twice IM on days 0 and 21 with PBS or the indicated vaccine combinations. Serum was collected by gingival bleeding on days 0, 14, 28, and 42. Syrian hamsters were infected intranasally with 1x10 ⁵ TCID50 live SARS-CoV-2 on day 45. Humoral immune responses were assessed based on (B) anti-S endpoint IgG titers and (C) wild-type SARS-CoV-2 neutralization titers. (D) Body weight changes (%) of hamsters were recorded daily after infection with SARS-CoV-2. (E) Virus titers in the lungs of hamsters infected with SARS-CoV-2 at 3 days post infection (dpi 3) were measured by TCID50 assay. (F) Representative histopathological images of H&E-stained lung sections of infected hamsters at 6 days post infection (dpi 6), where purple indicates inflamed areas. (G) Pathological severity of lung lesions was assessed as the percentage of inflamed area in each section. Symbols represent one animal; bars represent the geometric mean and 95% confidence interval of endpoint titer and ID50 titer in each group; horizontal lines represent mean and standard deviation (SD). For statistical analysis of antibody titer, viral titer, and pathological severity, significant differences between groups were determined by two-tailed Mann-Whitney test. Comparison of body weight change (%) was performed using two-way analysis of variance (ANOVA) with multiple comparison tests. * p ＜ 0.05, ** p ＜ 0.008, *** p ＜ 0.0005, **** p ＜ 0.0001, ns: not significant. Figure 6 is a set of figures showing the construction and characterization of pVax-based DNA vaccines. (A) Plasmid maps of pVax1 empty vector and (B) pVax-TS and (C) pVax-OVA, which express SARS-CoV-2 trimer-S (TS) and ovalbumin (OVA), respectively. (D) Agarose gel electrophoresis of plasmid DNA of pVax1, (E) pVax-TS and (F) pVax-OVA after restriction enzyme digestion. Figure 7 is a set of figures showing the validation of pVax-TS and pVax-OVA expression in transfected HEK293T cells. HEK293T cells were transfected with empty vector (pVax-1), SARS-CoV-2 trimer-S expression vector (pVax-TS), or rOVA expression vector (pVax-OVA). Cell lysates and culture supernatants were collected and analyzed by (A, C) SDS-PAGE and (B, D) immunoblotting with anti-SARS-CoV-2 spike antibody or anti-OVA antibody. Lane M: molecular weight ladder, relevant bands are marked in kilodaltons; Lanes 1 and 5: lysates of HEK293T cells transfected with pVax-1; Lanes 2 and 6: culture supernatants from HEK293T cells transfected with pVax-1; Lane 3: lysates of HEK293T cells transfected with pVax-TS; Lane 4: culture supernatants from HEK293T cells transfected with pVax-TS; Lane 7: lysates of HEK293T cells transfected with pVax-OVA; and Lane 4: culture supernatants from HEK293T cells transfected with pVax-OVA. FIG8 is a set of graphs showing a flow cytometric analysis flow chart to identify effector T cells in lymphocytes from immunized mice. Flow cytometric analysis of (A) lysed spleen cells including lymphocytes, (B) single cells in lymphocyte populations, (C) CD4+ T cells gated by CD3+ T cells, (D) T cells gated by lymphocytes (CD3+CD19-), (E) CD8+ T cells gated by CD3+ T cells, (F, G) effector T helper cells gated by CD4+ T cells (CD4+IFNγ+ or CD4+TNFα+), and (I, J) effector cytotoxic T cells gated by CD8+ T cells (CD8+IFNγ+ or CD8+TNFα+). Figure 9 is a set of graphs showing that the composite DNA+protein vaccine significantly increases the amount of effector CTLs. C57BL/6 mice (8/group) were immunized twice IM on days 0 and 21 with pVax-OVA (100 μg), rOVA/alum, or rOVA/alum combined with pVax-1 (100 μg). The PBS-injected group was used as a blank control. Spleens were collected from individual mice one week after the second vaccination. Splenocytes from each individual were cultured in medium as a control or restimulated with (A) OT-I epitope or (B) OT-II epitope. The number of IFN-γ-secreting cells was assessed by ELISpot. (C, D) The percentage of CD8+ T cells producing IFN-γ and TNF-α was analyzed by flow cytometry. (E, F) Vaccine-induced CTL killing assay in vivo. (E) Profiles of OT-I peptide (SII)-loaded target (CFSEhigh) and irrelevant peptide (RAH)-loaded control cells (CFSElow) in spleen were analyzed by flow cytometry. (F) The percentage of specific lysis was calculated by the following formula: Specific lysis % = [(% RAH peptide × A) - % SII peptide] / (% RAH peptide × A). Adjustment factor A = SII peptide from native control / RAH peptide. Each symbol represents a mouse; each bar represents the mean ± standard deviation of each group. Statistically significant differences were determined by a two-tailed Mann–Whitney test. * p ＜ 0.05, ** p ＜ 0.008, and *** p ＜ 0.0005 were considered significant; ns: not significant.

TWI843471B_112108585_SEQL.xml

Claims (9)

A composition, comprising: a single-unit vaccine, comprising a first dose of a single unit; and a nucleic acid vaccine, comprising a second dose of a vector; wherein the subunit of the single-unit vaccine comprises a recombinant SARS-CoV-2 trimerized spike (rTS) protein, and the vector of the nucleic acid vaccine is a plasmid encoding a SARS-CoV-2 trimerized spike (TS) sequence. The composition as described in claim 1, wherein the recombinant SARS-CoV-2 trimeric spike (rTS) protein includes a trimerization domain of IZN4. The composition as described in claim 1, wherein the subunit vaccine further comprises a third dose of an adjuvant. The composition as described in claim 3, wherein the adjuvant is an aluminum salt. The composition as described in claim 1, wherein the first dose is in the range of 0.1 μg to 10 μg. The composition as described in claim 1, wherein the second dose is in the range of 1 μg to 300 μg. The composition as described in claim 3, wherein the third dose is in the range of 100 μg to 500 μg. A use of a composition as described in any one of claims 1-7 for preparing a drug for preventing or ameliorating a disease, wherein the disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The use as described in claim 8, wherein the composition is in the range of 0.1 mg to 10 mg.

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