TW202202169A - Biodegradable nanocomplex vaccines, methods for prevention of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection - Google Patents

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. The protein or peptide derived from the S protein is a full-length receptor binding domain (residue 319-541 of the spike protein; SEQ ID NO:1).

Description

Biodegradable nanocomposite vaccine and method for preventing severe acute respiratory syndrome coronavirus type 2 (SARS-COV-2) infection

The present disclosure relates to vaccines for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), in particular to nanocomplex vaccines comprising SARS-CoV-2 antigens.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is considered to be the pathogen causing COVID-19 (Zhu, N. et al., "A Novel Coronavirus from Patients with Pneumonia in China," 2019.N. Engl. J. Med. 382, 727-733 (2020)). Compared with SARS-CoV, which broke out SARS in 2003, SARS-CoV-2 has relatively strong transmission ability. The report also pointed out that the clinical manifestations of COVID-19 are respiratory symptoms, and some patients may have severe cardiovascular damage. In addition, patients with underlying diseases, such as diabetes and cardiovascular disease (CVD), are at increased risk of death. The limited understanding of the pathogenic mechanism of SARS-CoV-2 limits the options for treatment options, however, vaccines can be developed rapidly due to their small relevance to the pathogenic mechanism.

In order to design a vaccine that can successfully target SARS-CoV-2, the following two points need to be considered: (1) The production of neutralizing antibodies: the vaccinated person is expected to produce neutralizing antibodies, and serum IgG is the most common resistance against virus transmission in the body However, for SARS-CoV-2 infected by the respiratory tract, secretory IgA plays the most important role; (2) Prevention of immunopathogenicity: Although the pathogenic mechanism of SARS-CoV-2 Still unknown, but reference can be made to several mechanisms defined in SARS-CoV. For example, direct viral effects and immunopathological factors contribute to diffuse alveolar damage (DAD) in SARS. In addition, in autopsy of SARS, infiltration of inflammatory cells composed of macrophages or a combination of macrophages and lymphocytes was observed regardless of the presence or absence of neutrophils. In other cases, a disproportionate lack of inflammatory cells has been noted. Excessive responses of the host immune system, especially dysregulation of proinflammatory cytokines and chemokines, may be responsible for severe SARS-related injury (Wong, CK et al., "Plasma inflammatory cytokines and chemokines in severe Acute respiratory syndrome," Clin. Exp. Immunol. 136, 95-103 (2004)). In addition, antibody-dependent enhancement (ADE) may be involved in SARS-CoV infection (Yip, MS et al., "Antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus," Virol.J .11, 82 (2014)). ADE illustrates enhanced viral infection through virus-receptor-mediated interactions mediated by sub-neutralizing antibodies (Flipse, J., Wilschut, J. & Smit, JM, "Molecular mechanisms involved in in antibody-dependent enhancement of dengue virus infection in humans.” Traffic 14, 25-35 (2013)).

Similar to other coronaviruses (CoV), SARS-CoV-2 is an enveloped, positive-stranded RNA virus with a spike protein composed of two subunits S1 and S2 (S protein). The homotrimer of the S protein forms the spines on the virus surface, and the S1 subunit binds to the host receptor-angiotensin-converting enzyme 2 (ACE2) (Yan, R. et al. , "Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2," Science (2020)). Previous studies have shown that the full-length S protein of SARS-CoV is highly immunogenic, but may also elicit deleterious immune responses in 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 effects (Wang, SF et al., "Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins,” Biochem. Biophys. Res. Commun. 451, 208-214 (2014)). In another alternative approach, the receptor binding domain (RBD) of the S protein has been suggested as a better vaccine candidate to avoid extreme immune responses and ADE (Jiang, S. et al., "Roadmap" to develop 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)).

Embodiments of the present disclosure use the nanocomposite vaccine technology described in US Patent No. US 10,052,390 B2, European Patent No. EP 2754436, Chinese Patent No. CN 103910892 B, and Taiwan Patent No. I511744. Briefly, by adding a charged polymer solution to another charged phase A simple electro-kinetic approach in counter-charged polymer solution to coat RBDs in nanocomposites. In these embodiments, the RBD is the encapsulated immunizing antigen in the first charged polymer solution.

The first charged polymer solution comprises immunogenic antigen (RBD) and negatively charged poly-γ-glutamic acid (γ-PGA) (preferably, weight average molecular weight (M.W.) of about 200 kDa or less). The second charged polymer solution contains positively charged chitosan (chitosan, CS). The weight-average molecular weight of chitosan is preferably about 10 to 100 kDa, suitable for proper solubilization at a pH that maintains the biological activity of protein and peptide drugs. Exemplary concentration ranges in these solutions are: RBD from 0.5 to 2 mg/mL, CS from 20 to 30 mg/mL, and γ-PGA from 5 to 20 mg/mL. Nanocomplexes (NCs) have zeta potentials of about +30 mV to about +50 mV and sizes ranging from about 100 nm to about 800 nm. These RBD-NCs are positively charged on the surface of the nanoparticles and have a unique ability to induce immune responses, thus possessing unique therapeutic effects in the prevention and treatment of SARS-CoV-2 infection.

One aspect of the present disclosure relates to a SARS-CoV-2 vaccine. According to one embodiment of the present disclosure, a SARS-CoV-2 vaccine comprises the SARS-CoV-2 receptor binding domain (RBD) of the spine (S) protein formulated in a nanocomplex. When an RBD is used as an immunizing antigen, it can be a separate peptide contained in a nanocomplex, or alternatively, it can form a fusion peptide with or without a linker in between. The nanocomposite contains gamma-polyglutamic acid (gamma-PGA) and chitosan, and any suitable concentrations of these components can be used in the embodiments of the present disclosure. For example, the concentration of RBD may be 0.5 to 2 mg/mL, the concentration of chitosan may be 20 to 30 mg/mL, and the concentration of γ-PGA may be 5 to 20 mg/mL. The nanocomposite can have a zeta potential of about +30 mV to about +50 mV.

Other aspects of the present disclosure will become apparent from the following description and drawings.

1A to 1C are schematic diagrams of the construction of SARS-CoV-2 RBD-His. Figure 1A shows a recombinant RBD containing residues 319 to 541 of the SARS-CoV-2 spine protein of the Wuhan-Hu-1 isolate, with the addition of an Igκ leader at the N-terminus of the RBD and a His ₆ tag at the C-terminus. Figure 1B shows sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified RBD-His protein, which is 5 micrograms of purified RBD-His protein subjected to SDS-PAGE and labeled with molecular weight (kDa ) mark. Figure 1C shows an RBD binding assay in which 1 microgram of purified RBD-His protein was incubated with 293T cells expressing hACE2 or DPP4 to test binding, where the right-shifted curve shows that RBD-His binds to hACE2 but not to DPP4 The curve and the gray shading representing binding to 293T cells are overlaid, which are marked as background. Representative graphs, percentage of positive cells and mean fluorescence intensity (MFI) are shown in the figures.

Figure 2 shows the Z mean, polydisperse index (PdI) and zeta potential of RBD protein or RBD-NC (nanocomplex). The polydispersity index (PdI) is determined by measurement of dynamic light scattering (DLS), where the polydispersity index is defined as the square of the standard deviation divided by the square of the mean.

Figure 3 shows a schedule of vaccination and blood sampling for antibody production assays in C57BL/6 or BALB/c mouse models using the vaccines of the present disclosure.

Figure 4 shows that the RBD-NC vaccine induces high titers of anti-RBD IgG antibodies in C57BL/6 mice vaccinated with NC only (NC), RBD-NC or RBD-Alum, respectively (n= 4-5). Mouse sera were collected at the indicated time points and titers of SARS-CoV-2 specific IgG were measured by ELISA. Data points represent the mean ± standard deviation (SD) of individual mice from two independent experiments; error bars reflect SD.

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

Figure 6 shows that serum from RBD-NC-vaccinated C57BL/6 mice is protective against SARS-CoV-2 infection in Vero E6 cell line.

Figure 7 shows that serum from BALB/c mice inoculated with RBD-NC is protective against SARS-CoV-2 infection in Vero E6 cell line.

Figure 8 shows a schematic representation of transduction of C57BL/6 mice via intratracheal and intraperitoneal injection of AAV/hACE2 and 3 subcutaneous inoculations of NC only or RBD-NC. Inoculated mice were challenged with 2 x 105 TCID ⁵⁰ of SARS-CoV-2 virus intranasally _at 10 weeks post-inoculation.

Figure 9 shows the viral load of SARS-CoV-2 in lung tissue after RBD-NC inoculation. Mice were sacrificed on day 5 post-infection, and the amount of virus in lung tissue was measured by TCID ₅₀ assay. Data points represent the mean ± standard deviation (SD) of individual mice in one experiment; error bars reflect SD.

Embodiments of the present disclosure relate to SARS-CoV-2 vaccines that can be used to prevent SARS-CoV-2 infection. The vaccines of the present disclosure contain antigens in unique nanocomplexes that can elicit an effective immune response. The present disclosure prepares nanoparticle-based vaccines by an electro-kinetic method, which is very different from general vaccine technology. The technology operates a solution system of Electric double layer to coat proteins with (+/-) charged polymers by compressive force to form stable, narrow charge distribution and dispersed spherical nanocomplexes (see US Patent No. US 10,052,390 B2; European Patent No. EP 2754436; Chinese Patent No. CN 103910892 B; Taiwan Patent No. I511744).

The vaccine of the present disclosure can use commercially available SARS-CoV-2 antigenic proteins or recombinant proteins or fragments thereof. The recombinant RBD contains residues 319 to 541 of the SARS-CoV-2 spine protein of Wuhan-Hu-1 isolate. Figure 1A shows a construct used to generate RBD antigens containing an IgK leader peptide at the N-terminus and a His ₆ tag at the C-terminus. Figure 1B shows the results of analysis of the expressed recombinant RBD protein by polyacrylamide gel electrophoresis (SDS-PAGE), showing a substantially clear band at a molecular weight of about 30 kDa.

To confirm the binding affinity of purified RBD to the native receptor, 1 microgram of purified RBD-His protein was incubated with hACE2-expressing 293T cells or DPP4-expressing 293T cells. As shown in Figure 1C, the curve shifted to the right represents RBD-His binding to hACE2, while the curve bound to DPP4 overlaps the gray shaded area representing binding to 293T cells (background control). A representative graph, percentage of positive cells (82%) and mean fluorescence intensity (MFI) are shown in Figure 1C. These results clearly indicate that recombinant RBD is functional and can specifically bind to hACE2. This antigen is used to test the vaccines of the present disclosure.

To prepare antigen-nanocomplexes, SARS-CoV-2 RBD was mixed with γ-PGA to form a first charged polymer solution. This solution is then mixed with a second charged polymer solution (eg, chitosan) in appropriate proportions. The resulting RBD-nanocomplexes (RBD-NCs) were characterized by dynamic light scattering (DLS). The results of DLS showed that the size of RBD-NCs ranged from about 100 nm to about 800 nm (Figure 2). Low polydispersity index as determined by dynamic light scattering (DLS) (polydispersity index, PdI) demonstrated that the size range of the NC particles was not large, and the zeta potential of these NCs ranged from about +30 mV to about +50 mV (Figure 2).

Next, these antigen-NCs were tested for their ability to elicit an immune response. RBD-NC and RBD-Alum (as a general adjuvant control group) were tested at 10 μg per dose to evaluate the stimulation of antibody production. The control group was NC only (negative control group without antigen). C57BL/6 or BALB/c mice were vaccinated by the subcutaneous (S.C.) route at weeks 0, 2, and 6, and blood samples were collected at weeks 0, 2, and 4 (Figure 3).

C57BL/6 mice (Fig. 4) and BALB/c mice (Fig. 5) vaccinated with RBD-NC produced high titers of anti-RBD antibodies two weeks after the first inoculation and after the second boost injection 10- to 40-fold higher titers of anti-RBD antibodies were then produced. In contrast, RBD-Alum could hardly induce the production of anti-RBD antibodies after the first injection, and the antibody titers produced after the second injection were 100-fold lower than that of RBD-NC. These results show that RBD-NC has an unexpected effect as a vaccine, which can effectively stimulate the immune response and generate high titers of anti-RBD antibodies.

Neutralization assays were performed to test whether RBD-NCs could induce neutralizing antibodies. Serum from vaccinated mice was collected at 4 weeks after the second vaccination and incubated with SARS-CoV-2 virus at different dilutions. The mixture was then co-cultured with Vero E6 cells to see if the inoculated mouse serum could protect the cells from viral infection. As shown in Figure 6 and Figure 7, after inoculation with RBD-NC vaccine, mouse sera from C57BL/6 (Figure 6) and BALB/c (Figure 7) mice produced high titers of neutralizing antibodies, which were tested in cell experiments. Inhibition of SARS-CoV-2 virus infection.

To further test the potential of RBD-NC to protect humans from SARS-CoV-2 infection, animal models were used to study the effects of these vaccines, that is, by inoculating RBD-NC into Prof. Tao Mihua (Biomedical Research Institute of Academia Sinica, Taiwan). in an animal model transduced with AAV/hACE2 developed by Briefly, C57BL/6 mice were transduced by intratracheal and intraperitoneal injection of AAV/hACE2. Then, AAV-transduced mice were inoculated 3 times with NC alone or RBD-NC, and 2 × 10 ⁵ median tissue culture infectious dose (median tissue culture infectious dose) in the nasal cavity 10 weeks after the first inoculation was performed. dose, _TCID50 ) of SARS-CoV-2 virus challenged vaccinated mice (Figure 8).

Mice vaccinated with RBD-NC after AAV/hACE2 transduction were challenged with SARS-CoV-2 virus by intranasal injection. As shown in Figure 9, on day 5 post-infection, all control mice showed high titers of viral load in lung tissue. In contrast, all mice vaccinated with RBD-NC had undetectable viral titers and were largely protected from SARS-CoV-2 infection. These results show that RBD-NC can induce the production of antibodies in animals, and the antibodies produced can protect animals from SARS-CoV-2 infection.

Embodiments of the present disclosure will be further illustrated by the following specific examples. Those of ordinary skill in the art will understand that these examples are for illustration only and are not meant to limit the scope of the present disclosure as other modifications and changes are possible.

Example

1. Purification of RBD

The RBD-His protein was produced in the ExpiCHO Expression System (Thermo Scientific, A29133) and purified by the ProBond Purification System (Novex, K850-01). Briefly, supernatants were collected 10 days after ExpiCHO cells were transfected with pcDNA3-Igκ-RBD-His and subjected to protein purification on Ni columns.

2. Combination test

To measure the binding ability of SARS-CoV-2 RBD-His protein to different receptors, 293T cells were transfected with hACE2 or DPP4 using Lipofectamine 2000 (ThermoFisher, 11668500) for 2 days. Then, cells were detached from the culture dish and incubated with 1 μg of purified RBD-His protein in 100 μL of staining buffer (1% FBS in DPBS) for 1 hour. After washing away unbound proteins, cells were incubated with 0.5 μg PE-conjugated goat anti-mouse IgG-Fc (Jackson 115-116-146) in 100 μL staining buffer for 30 minutes. Non-viable cells were excluded using 7-amino-actinomycin D (7-AAD, Biolegend, 420404). Stained cells were analyzed using FACSCanto (BD Biosciences) and data processed using FlowJo V10 software.

3. Preparation and characterization only NC, RBD-NC and RBD-Alum

A first solution was prepared with γ-polyglutamic acid (γ-PGA; w/v in ddH2O = ₁ %; weight average molecular weight range = about 200 kDa or less) and with/without a predetermined amount of RBD. A second solution was prepared with chitosan in 1% acetic acid (w/v = 2.5% chitosan, weight average molecular weight range = about 10 to 100 kDa). The second solution (chitosan solution) was added to the first solution (γ-PGA with RBD) to form nanocomplexes (NC). NCs were stored overnight at 4°C for stability testing, and their size, zeta potential and polydispersity index (PdI) were determined with a Malvern Zetasizer Nano series (Zetasizer Nano ZS, Malvern Panalytical Ltd., UK). 10 micrograms of purified RBD-His protein were added to aluminum hydroxide (Thermo, 77161) and mixing continued for 1 hour.

4. Mice

All animal studies were performed under specific conditions free of pathogens. In the antibody induction experiments, 6- to 8-week-old female C57BL/6 or BALB/c mice purchased from the National Laboratory Animal Center in Taiwan were divided into 3 groups: NC only (5 mice), 10 μg/dose RBD-Alum (5 mice) and 10 μg/ Dose RBD-NC (5 mice). Mice were vaccinated with these vaccines at weeks 0, 2 and 6 by the subcutaneous route. Serum was collected every 2 weeks for SARS-CoV-2 RBD-specific antibody responses.

In a mouse model expressing human ACE2, 6- to 8-week-old female C57BL/6 mice were purchased from the National Laboratory Animal Breeding and Research Center in Taiwan. To establish durable ACE2 gene expression in the lungs of immunocompetent mice, we used AAV serotype 6 vector (AAV serotype 6 vector, AAV6) and AAV serotype 9 vector (AAV serotype 9 vector) with high lung transduction rates , AAV9) to deliver the human ACE2 gene. Mice were intratracheally administered 3 x 10 ¹¹ AAV6/ACE2 suspended in 100 μL of physiological saline and 1 x 10 ¹² AAV9/ACE2 suspended in 100 μL of physiological saline intraperitoneally. Subsequently, the magnitude of ACE2 expression in the lungs was measured.

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

To detect SARS-CoV-2 RBD-specific antibodies in the serum of vaccinated mice, ELISA plates were coated with SARS-CoV-2 RBD-His protein at a final concentration of 5 μg/mL in carbonate coating buffer (Thermo Scientific, 430341). The plates were then washed with PBS and blocked with 3% skim milk for 2 hours. Serially diluted mouse serum was added and incubated for one hour at room temperature. HRP-conjugated goat anti-mouse IgG Fc antibody (Chemicon, AP127P) was diluted 1/1,000 in blocking solution for detection. The discs were then developed with TMB (BD, 555214) for 10 minutes and the reaction was stopped with 2 NH ₂ SO ₄ . Absorbance values were measured at 450 nm in a microplate reader.

6. Neutralization test for SARS-CoV-2

To detect neutralizing antibodies against live SARS-CoV-2 in vaccinated mice, mouse sera were collected 6 weeks after the first vaccination and serially diluted 2-fold from 20-fold. Diluted mouse serum was pre-incubated with 100 TCID ₅₀ of live SARS-CoV-2 virus for one hour before transferring into Vero E6 cells. Three days after infection, the cytopathic effect (CPE) was recorded under the microscope, and the highest serum dilution concentration that completely inhibited virus-induced CPE was determined as the neutralization titer.

7. Viral titers in lung tissue

Virus titers in lung tissue were measured by TCID ₅₀ assay. Briefly, lung tissue from challenged mice was collected on day 5 post-infection and homogenized with a SpeedMill homogenizer (Analytik Jena). Ten-fold serial dilutions of the homogenate were incubated with Vero E6 cells at 37°C for 3 to 5 days. The virus titer was taken as the dilution factor that would give rise to CPE in 50% of the cells.

Embodiments of the present disclosure have been described with a limited number of examples. Those skilled in the art will appreciate that other modifications and changes are possible without departing from the scope of the present disclosure. Therefore, the claimed scope of the present disclosure should be limited only by the scope of the appended claims.

<110> Academia Sinica British Cayman Islands Business Prophet Biotechnology Co., Ltd.

<120> Biodegradable nanocomposite vaccine and method for preventing severe acute respiratory syndrome coronavirus type 2 (SARS-COV-2) infection

<150> US 63/004493

<151> 2020-04-02

<160> 1

<210> 1

<211> 223

<212> PRT

<213> Coronaviridae

<400> 1

Claims (5)

A nanocomplex vaccine for generating immunity against SARS-CoV-2 (2019-nCoV) infection, the nanocomplex vaccine is composed of proteins or peptides derived from the SARS-CoV-2 spike protein , wherein the protein or peptide comprises the receptor binding domain of the spike protein and is encapsulated in a nanocomplex. The nanocomplex vaccine of claim 1, wherein the protein or peptide is residues 319 to 541 of the full-length receptor binding domain (SEQ ID NO: 1). The nanocomplex vaccine of claim 2, wherein the nanocomplex is formed with poly-γ-glutamic acid and chitosan. The nanocomplex vaccine of claim 3, wherein the poly-γ-glutamic acid has a weight average molecular weight of about 200 kDa or less, and the chitosan has a weight average molecular weight of about 10 to 100 kDa. The nanocomplex vaccine of claim 3, wherein the nanocomplex has a zeta potential ranging from about +30 mV to about +50 mV and a size ranging from about 100 nm to about 800 nm.

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