WO2021203018A1

WO2021203018A1 - Biodegradable nanocomplex vaccines, methods for prevention of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection

Info

Publication number: WO2021203018A1
Application number: PCT/US2021/025608
Authority: WO
Inventors: Haishan JANG; Ping-Yen Huang; Yu-Hung Chen; Yan-wei WU; Mi-Hua Tao; I-Jung Lee; Lan YU-HUA; Cheng-pu SUN
Original assignee: Ascendo Biotechnology, Inc.; Academia Sinica
Priority date: 2020-04-02
Filing date: 2021-04-02
Publication date: 2021-10-07
Also published as: WO2021203017A3; TW202202169A; WO2021203017A2

Abstract

A nanocomplex vaccine for generation of immunity against SARS-CoV-2 (2019-nCoV) infection includes a protein or peptide derived from SARS-CoV-2 encapsulated in a nanocomplex, wherein the protein or peptide derived from the SARS-CoV-2 is a full-length receptor binding domain of the spike protein (residue 319-541 of spike protein; SEQ ID NO:1).

Description

BIODEGRADABLE NANOCOMPLEX VACCINES, METHODS FOR PREVENTION

OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-CoV-2)

INFECTION

FIELD OF INVENTION

[0001] The invention relates to severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) vaccines, particularly to nanocomplex vaccines comprising a SARS-CoV-2 antigen.

BACKGROUND

[0002] The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the pathogen causing COVID-19. Compared with the SARS-CoV that caused an outbreak of SARS in 2003, SARS-CoV-2 has a relatively stronger transmission capacity. The clinical manifestations of COVID-19 are reported by respiratory symptoms and some patients may have severe cardiovascular damage. In addition, there is an increasing risk of death in patients with underlying diseases, such as diabetes and cardiovascular diseases (CVDs). Limited understanding of the pathogenesis of SARS-CoV-2 restricts the treatments; however, vaccine can be developed quickly because it is less related to the pathogenesis.

[0003] To design a successful vaccine against SARS-CoV-2, two points need to be considered. (1) Production of neutralizing antibody: People vaccinated will be expected to produce neutralizing antibodies. The serum IgG is the most common neutralizing antibodies against virus spread in body. However, in the case of SARS-CoV-2, which infects through the respiratory route, the secretory IgA plays the most important role. (2) Prevention of immunopathogenetic: Although the pathogenesis of SARS-CoV-2 is still unknown, several mechanisms defined in SARS-CoV may be referenced. The diffuse alveolar damage (DAD) in SARS caused both by direct viral effects and immunopathogenetic factors. Inflammatory cells infiltration that consists of macrophages or a combination of macrophages and lymphocytes with or without neutrophils were observed in SARS autopsies. In other cases, a disproportionate scarcity of inflammatory cells has been noted. An excessive reaction of the host’s immune system, particularly dysregulation of proinflammatory cytokines and chemokines may be the reason for severe SARS-related injuries. Furthermore, antibody-dependent enhancement (ADE) may play a role in SARS-CoV infection (Yip, M.S. et al., “ Antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus f Virol J 11, 82 (2014)). ADE describes an enhanced virus infection through sub-neutralizing antibodies mediating interactions between virus and receptor (Flipse, J., Wilschut, J. & Smit, J.M., “ Molecular mechanisms involved in antibody-dependent enhancement of dengue virus infection in humans ” Traffic 14, 25-35 (2013)).

[0004] SARS-CoV-2 is an enveloped, positive-stranded RNA virus like other coronaviruses (CoVs). They have spike glycoproteins (S proteins) composed of two subunits, SI and S2. Homotrimers of S proteins form the spikes on the viral surface, and the S 1 subunit binds to host receptors, angiotensin-converting enzyme 2 (ACE2) (Yan, R. et al., “ Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2f Science (2020)).

[0005] Previous studies demonstrated that full-length S protein of SARS-CoV has high immunogenicity but may also induce harmful immune responses in the vaccinated animals (Czub, M., Weingartl, H , Czub, S., He, R. & Cao, J., “ Evaluation of modified vaccinia virus Ankara based recombinant SARS vaccine in ferrets ,” Vaccine 23, 2273-2279 (2005)) or ADE effect (Wang, S.F. et al., “ Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins ,” Biochem. Biophys. Res. Commun. 451, 208-214 (2014)). In an alternative approach, the receptor binding domain (RBD) of the S protein is recommended as a better vaccine candidate to avoid extreme immune response and ADE (Jiang, S. et al., “ Roadmap to developing a recombinant coronavirus S protein receptor-binding domain vaccine for severe acute respiratory syndrome ,” Expert. Rev. Vaccines 11, 1405-1413 (2012); Wang, Q. et al., “ Immunodominant SARS Coronavirus Epitopes in Humans Elicited both Enhancing and Neutralizing Effects on Infection in Non-human Primates ,” ACS Infect Dis. 2, 361-376 (2016)).

SUMMARY

[0006] Embodiments of the invention use nanocomplex vaccine technology described in U.S. PatentNo. 10,052,390 B2, EP 2754436, Chinese Patent No. CN103910892B, and Taiwan Patent No. 1511744. Briefly, RBD is encapsulated in nanocomplexes using a simple electro-kinetic approach by addition of a charged polymer solution into another oppositely charged polymer solution. In these embodiments, RBD is the encapsulated immunogen in the first charged polymer solution.

[0007] The first charged polymer solution contains the immunogen (RBD) and poly-g- glutamic acid (γ-PGA) (weight-averaged M.W. preferably about 200 kDa or less) having negative charges. The second charged polymer solution contains chitosan (CS) having positive charges. The weight-averaged molecular weight (MW) of chitosan is preferably about 10-100 kDa, adapted for adequate solubility at a pH that maintains the bioactivity of protein and peptide drugs. The exemplary ranges of concentrations in these solutions are: RBD: 0.5 to 2 mg/ml, CS: 20 to 30 mg/ml, and g-PGA: 5-20 mg/ml. The nanocomplexes (NCs) have zeta potentials of from about +30 mV to about +50 mV and a size range from about 100 nm to about 800 nm. These RBD-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.

[0008] One aspect of the invention relates to SARS-CoV-2 vaccines. A SARS-CoV-2 vaccine according to one embodiment of the invention comprises SARS-CoV-2 receptor binding domain (RBD) of the spike (S) protein formulated in nanocomplexes. When RBD is used as immunogen, it can be separate peptides contained in the nanocomplexes, or alternatively they may form a fusion peptide with or without a linker therebetween. The nanocomplexes comprise g- polyglutamic acid (g-PGA) and chitosan. Any suitable concentrations of these components may be used with embodiments of the invention. For example, a concentration of RBD may be 0.5 to 2 mg/ml, a concentration of chitosan may be 20 to 30 mg/ml, and a concentration of g-PGA may be 5-20 mg/ml. The nanocomplexes may have a zeta potential of about +30 mV to about +50 mV.

[0009] Other aspects of the invention would become apparent with the following description and the accompanying drawings.

BRIEF DESCROPTION OF THE DRAWINGS

[0010] FIG. 1 shows the schematic construction of SARS-CoV-2 RBD-His. (A) The recombinant RBD comprised residues 319-541 of SARS-CoV-2 spike of Wuhan-Hu-1 isolate. The IgK leader was added at the N-terminus and a His₆ tag at the C-terminus of the RBD. (B) SDS-PAGE of purified RBD-His protein. Five micrograms of purified RBD-His protein were subjected to SDS-PAGE. The molecular weight marker (kDa) was indicated. (C) RBD binding assay. One microgram of purified RBD-His protein was incubated with either hACE2- or DPP4- expressing 293 T cells to test the binding capacity. The right-shift curve showed the binding of RBD-His with hACE2, while the curve for binding with DPP4 overlaps the gray shading representing binding with 293T cells, marked as background. Representative image, positive cell percentage and MFI were shown.

[0011] FIG. 2 shows the Z-average, polydiseperse index (Pdl) and zeta-potential of RBD protein or RBD-NCs (nanocomplexes). Pdl (Polydisepersity Index) is determined by DLS (Dynamic light scattering) measurements. PDI is defined as the square of the standard deviation divided by the square of the mean.

[0012] FIG. 3 shows a schedule of vaccinations and blood samplings using C57BL/6 or BALB/c mice models for antibodies production testing using the vaccines of the invention. [0013] FIG. 4 shows that the RBD-NCs vaccine induces high titers of anti-RBD IgG antibodies in C57BL/6 mice. C57BL/6 mice were immunized with either NCs only, RBD-NCs, or RBD-Alum (n=4-5). Mouse sera were collected at the indicated time points and SARS-CoV- 2-specific IgG titers were measured with ELISA. Data points represent mean ± SD of individual mice from two independent experiments; error bars reflect SD.

[0014] FIG. 5 shows that the RBD-NCs vaccine induces high titers of anti-RBD IgG antibodies in BALB/c mice. BALB/c mice were immunized with either NCs only, RBD-NCs, or RBD-Alum (n=4-5). Mouse sera were collected at the indicated time points and SARS-CoV- 2-specific IgG titers were measured with ELISA. Data points represent mean ± SD of individual mice from two independent experiments; error bars reflect SD.

[0015] FIG. 6 shows that sera from the RBD-NCs vaccinated C57BL/6 mice have protective effect against SARS-CoV-2 infection in Vero E6 cell line.

[0016] FIG. 7 shows that sera from the RBD-NCs vaccinated BALB/c mice have protective effect against SARS-CoV-2 infection in Vero E6 cell line.

[0017] FIG. 8 shows a schematic diagram that C57BL/6 mice were transduced with AAV/hACE2 through intratracheal and intraperitoneal injection and immunized with NCs only or RBD-NCs subcutaneously three times. The vaccinated mice were then challenged with 2 x 10⁵ TCID50 of SARS-CoV-2 virus intranasally at week 10 after immunization.

[0018] FIG. 9 shows the SARS-CoV-2 viral loads in lung tissues after the vaccination of RBD-NCs. Mice were sacrificed at day 5 after infection, and the viral loads in lung tissues were measured with TCID₅₀ assay. Data points represent mean ± SD of individual mice from one experiment; error bars reflect SD.

DETAILED DESCRIPTION [0019] Embodiments of the invention relate to SARS-CoV-2 vaccines that can be used in the prevention for SARS-CoV-2 infections. A vaccine of the invention comprises an antigen in unique nanocomplexes that can elicit effective immune responses. Inventors of the invention found an electro-kinetic approach to preparing nanoparticle-based vaccine. This approach is very 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 nanocomplex (cf. U.S. Patent No. 10,052,390 B2; EU: 2754436; China: CN103910892B; Taiwan: 1511744).

[0020] Vaccines of the invention may use commercially available SARS-CoV-2 antigen proteins or recombinant proteins or fragments thereof. The recombinant RBD comprised residues 319-541 of SARS-CoV-2 spike of Wuhan-Hu-1 isolate. A construct for production of a RBD antigen is illustrated in FIG. 1A, which also includes an IgK leader peptide at the N- terminus and a His₆ tag at the C-terminus. Results of SDS-PAGE analysis of the expressed recombinant RBD protein are shown in FIG. IB, showing a substantially clean band with a molecular weight around 30 kDa.

[0021] To confirm the binding affinity of purified RBD to nature receptors, one microgram of purified RBD-His protein was incubated with either hACE2-expressing 293T cells or DPP4- expressing 293T cells. As shown in FIG. 1C, the right shifted curve represents the binding of RBD-His with hACE2, while the curve for binding with DPP4 overlaps with the gray shading area that represents binding with 293T cells (background control). Representative image, percentage of positive cells (82%) and mean fluorescence intensity (MFI) are shown in FIG. 1C. These results clearly show that the recombinant RBD is functional and can bind specifically with hACE2. This antigen was used to test vaccines of the invention. [0022] To prepare antigen-nanocomplexes, the SARS-CoV-2 RBD was mixed with g-PGA to form a first charged polymer solution. Then, this solution was mixed with a second charged polymer solution (e.g., chitosan) in an appropriate ratio. The resulting RBD-nanocomplexes (RBD-NCs) were characterized with dynamic light scattering (DLS). Results of DLS show the sizes of RBD-NCs range from about 100 nm to about 800 nm (FIG. 2). The NC particles do not have a wide range of particle size variations, as evidenced by the low polydispersity index (Pdl) determined by dynamic light scattering (DLS), and the zeta-potentials of these NCs were determined to range from about +30 mV to about +50 mV (FIG. 2).

[0023] These antigen-NCs were tested for their abilities to elicit immune responses. The RBD-NCs and RBD-Alum (as a conventional adjuvant control) were tested at 10 pg per dose to assess the stimulation of antibody productions. The control is NCs only (a negative control without an antigen). The vaccines were inoculated into C57BL/6 or BALB/c mice through subcutaneous (S.C.) routes at weeks 0, 2 and 6. The blood samples were obtained at weeks 0, 2 and 4 (FIG. 3).

[0024] Both C57BL/6 (FIG. 4) and BALB/c (FIG. 5) mice that were immunized with RBD- NCs generated high titers of anti-RBD antibody at two weeks after first immunization and produced 10 to 40 folds higher titers of anti-RBD antibodies after the second (boost) injection. In contrast, the Alum-RBD could barely induce anti-RBD antibody productions after first immunization and generated a hundred-fold lower antibody titers after the second injection, as comparing with RBD-NCs. These results show the unexpected efficacies of the RBD-NCs as vaccines.

[0025] The neutralization assay was conducted to examine whether RBD-NCs could induce neutralizing antibodies. Sera from the vaccinated mice were collected at week 4 after the second immunization and incubated with the SARS-CoV-2 virus with different dilution folds. The mixtures were then co-cultured with Vero E6 cells to see if the vaccinated mouse serum could protect virus from infection. As shown in FIG. 6-7, mouse sera from both C57BL/6 (FIG. 6) and BALB/c (FIG. 7) mice after RBD-NCs vaccination generated high titers of neutralizing antibodies and prevented SARS-CoV-2 virus infection in vitro.

[0026] To further examine the potential of RBD-NCs to protect SARS-CoV-2 infection in human, we investigated the effects of these vaccines using an animal model - i.e., by inoculating RBD-NCs into AAV/hACE2 transducing animal model, which were generated by Prof. Mi-Hua Tao (Institute of Biomedical Sciences, Academia Sinica, Taiwan). In brief, C57BL/6 mice were transduced with AAV/hACE2 through intratracheal and intraperitoneal injections. The AAV transduced mice were then immunized with NCs only or RBD-NCs three times and challenged with 2 x 10⁵ Median Tissue Culture Infectious Dose (TCID50) of SARS-CoV-2 virus intranasally at 10 weeks after the first immunization (FIG. 8).

[0027] The AAV/hACE2 transduced and then RBD-NCs vaccinated mice were challenged with SARS-CoV-2 virus through intranasal infection. As shown in FIG. 9, all controlled mice showed high titers of viral loads in lung tissues at day 5 after the infection. By contrast, all RBD- NCs vaccinated mice were largely protected against SARS-CoV-2 infections with undetectable viral titers. These results show that RBD-NCs can induce the production of antibodies in the animals and that the produced antibodies can protect the animals from SARS-COV-2 infections.

[0028] Embodiments of the invention will be further illustrated with the following specific examples. One skilled in the art would appreciate that these examples are for illustration only and are not meant to limit the scope of the invention because other modifications and variations are possible.

EXAMPLES

1. RBD purification.

[0029] The RBD-His protein was produced in ExpiCHO expression system (Thermo Scientific, A29133) and purified by ProBond Purification system (Novex, K850-01). In brief, ExpiCHO cells were transfected with pcDNA3-IgK-RBD-His for 10 days. The supernatant was then collected and applied to a Ni column for protein purification.

2. Binding assay.

[0030] For measuring the binding capacity of SARS-CoV-2 RBD-His protein with different receptors, 293 T cells were transfected with either hACE2 or DPP4 using lipofectamine 2000 (Therm oFisher, 11668500) for 2 days. Cells were then dissociated from the culture plates and incubated with 1 μg of purified RBD-His protein in 100 pi staining buffer (1 % FBS in DPBS) for an hour. After washing out the non-binding protein, cells were then incubated with 0.5 pg of PE-conjugated goat anti-mouse IgG-Fc (Jackson 115-116-146) in 100 mΐ staining buffer for 30 minutes. 7-amino-actinomycin D (7-AAD) (Biolegend, 420404) was used to exclude the non- viable cells. The stained cells were analyzed using FACSCanto (BD Biosciences) and data were processed using FlowJo VI 0 software.

3. The preparation and characterization of NCs only, RBD-NCs, and RBD-Alum.

[0031] A first solution is prepared with g-Polyglutamic acid (γ-PGA; w/v=l% in ddH O; weight-averaged M.W. range = about 200 kDa or less) and with/without a predetermined amount of RBD. 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 RBD) 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.). Ten micrograms of purified RBD-His protein were adding to aluminum hydroxide (Thermo, 77161) and continued mixing for an hour.

4. Mice.

[0032] All animal studies were conducted under specific pathogen-free conditions. In antibody induction experiments, six- to eight-week-old female C57BL/6 or BALB/c mice, purchased from National Laboratory Animal Center, Taiwan, were divided into 3 groups: NC only (5 mice), 10 pg/dose RBD-Alum (5 mice), and 10 pg/dose RBD-NC (5 mice). Mice were inoculated with these vaccines at weeks 0, 2 and 6 through subcutaneous routes. Sera were collected every 2 weeks to detect SARS-CoV-2 RBD-specific antibody responses.

[0033] In human ACE2-expressed mice model, six- to eight-week-old female C57BL/6 mice were purchased from National Laboratory Animal Breeding and Research Center, Taiwan. To establish persistent ACE2 gene expression in the lung of immunocompetent mice, we used the AAV serotype 6 vector (AAV6), which has a high lung transduction rate, and AAV serotype 9 vector (AAV9) to deliver the human ACE2 gene. Mice are intratracheal administrated with 3 x 10¹¹ AAV6/ACE2-suspended in 100 pi saline and intraperitoneal administrated with 1 x 10¹² AAV9/ACE2-suspended in 100 pi saline. Subsequently, lung ACE2 expression levels are measured.

5. Enzyme-linked immunosorbent assay (ELISA) for anti-RBD antibodies detection.

[0034] For detecting SARS-CoV-2 RBD-specific antibodies in vaccinated mouse sera, plates (Thermo Scientific, 430341) were coated with SARS-CoV-2 RBD-His protein in carbonate coating buffer at a final concentration of 5 pg/ml. Plates were then washed with PBS and blocking with 3% skim milk for two hours. Serially diluted mouse sera were added and incubated at room temperature for an hour. HRP-conjugated goat anti-mouse IgG Fc antibody (Chemicon, AP127P) was diluted 1/1000 in blocking solution for detection. The plates were then developed with TMB (BD, 555214) for 10 minutes and stopped with 2N H2SO4. The absorbance was measured in a microplate reader at 450 nm.

6. Neutralization assay of SARS-CoV-2.

[0035] For detecting neutralizing antibodies against live SARS-CoV-2 in the vaccinated mice, mouse sera were collected at week 6 after first immunization and performed a serial 2-fold dilutions from 20-fold. The diluted mouse sera were pre-incubated with 100 TCID50 of live SARS-CoV-2 virus for an hour and then transferred to Vero E6 cells. Three days after infection, the cytopathic effect (CPE) was recorded under microscope and the neutralizing titer was determined as the highest dilution of sera that completely inhibited virus-induced CPE.

7. Viral titers in lung tissue.

[0036] Viral titer in lung tissue was measured by TCID₅o assay. Briefly, lung tissue from the challenged mice at day 5 post infection was collected and homogenized by the SpeedMill homogemzer (Analytik Jena). Ten-fold serially diluted homogenates were then incubated with Vero E6 cells at 37°C for 3-5 days. The dilution fold that caused CPE in 50% of the cells was determined as the viral titer.

[0037] Embodiments of the invention have been described with a limited number of examples. One of 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 be limited only by the attached claims.

Claims

CLAIMS What is claimed is:

1. A nanocomplex vaccine for generation of immunity against SARS-CoV-2 (2019-nCoV) infection, consisting of a protein or peptide derived from spike protein of SARS-CoV-2, wherein the protein or peptide contains the receptor-binding domain (RBD) of the spike protein and is encapsulated in a nanocomplex.

2. The nanocomplex vaccine according to claim 1, wherein the protein or peptide is the full- length receptor binding domain (residue 319-541) SEQ ID NO: 1.

3. The nanocomplex vaccine according to claim 2, wherein the nanocomplex is formed with poly-γ-glutamic acid (g-PGA) and chitosan (CS).

4. The nanocomplex vaccine according to claim 3, wherein the poly-γ-glutamic acid (g-PGA) has a weight-averaged molecular weight (MW) of about 200 kDa or less and the chitosan (CS) has a weight-averaged molecular weight (MW) of about 10-100 kDa.

5. The nanocomplex vaccine according to claim 3, wherein the nanocomplex has a zeta potential of from about +30 mV to about +50 mV and a size range from about 100 nm to about 800 nm.