ABSTRACT
SARS-CoV-2 is a particularly transmissible virus that renders the worldwide COVID-19 pandemic and global severe respiratory distress syndrome. Protein-based vaccines hold great advantages to build the herd immunity for their specificity, effectiveness, and safety. Receptor-binding domain (RBD) of SARS-CoV-2 is an appealing antigen for vaccine development. However, adjuvants and delivery system are necessitated to enhance the immunogenicity of RBD. In the present study, RBD was chemically conjugated with loxoribine and SpyCatcher/SpyTag, followed by assembly to form a nanoparticle vaccine. Loxoribine (a TLR7/8 agonist) acted as an adjuvant, and nanoparticles functioned as delivery system for the antigen and the adjuvant. The nanoparticle vaccine elicited high RBD-specific antibody titers, high neutralizing antibody titer, and strong ACE2-blocking activity. It stimulated high splenic levels of Th1-type cytokines (IFN-γ and IL-2) and Th2-type cytokines (IL-4 and IL-5) in BALB/c mice. It promoted the splenocyte proliferation, enhanced the CD4+ and CD8+ T cell percentage and stimulated the maturation of dendritic cells. The vaccine did not render apparent toxicity to the organs of mice. Thus, the nanoparticle vaccine was of potential to act as a preliminarily safe and effective candidate against SARS-CoV-2.
Subject(s)
COVID-19 , Nanoparticles , Animals , Humans , Mice , COVID-19 Vaccines , SARS-CoV-2 , COVID-19/prevention & control , Pandemics , Adjuvants, Immunologic , Adjuvants, Pharmaceutic , Cytokines , Mice, Inbred BALB C , Antibodies, Neutralizing , Antibodies, ViralABSTRACT
SARS-CoV-2 is a respiratory virus that causes significant threats to human health. Mucosal immunity provides a first-line defense to prevent the infection of SARS-CoV-2 in the respiratory tract. Because most SARS-CoV-2 vaccines could not stimulate mucosal immunity in the respiratory tract, appropriate mucosal adjuvants or delivery systems are needed for mucosal vaccine development. Mannan, polyarginine, and 2',3'-cGAMP are three mucosal adjuvants that could stimulate mucosal immunity. In the present study, the three adjuvants were assembled with a receptor-binding domain (RBD) by electrostatic interaction to generate a nanoparticle vaccine (RBD-MP-cG). RBD-MP-cG elicited mucosal IgA and IgG response in bronchoalveolar lavage and nasal lavage by intranasal administration. It induced a robust RBD-specific antibody response, high levels of protective neutralizing antibody, and ACE2-blocking activity in the mouse sera. It stimulated the splenic secretion of high levels of Th1-, Th2-, and Th17-type cytokines. Thus, RBD-MP-cG elicited strong mucosal immunity and systematic immunity by intranasal administration. RBD-MP-cG was expected to act as a safe, effective, and easily produced mucosal nanoparticle vaccine to combat the infection of SARS-CoV-2.
Subject(s)
COVID-19 , Nanoparticles , Humans , Animals , Mice , COVID-19 Vaccines , SARS-CoV-2 , Antibodies, Viral , Administration, Intranasal , COVID-19/prevention & control , Adjuvants, Immunologic/pharmacology , Adjuvants, Pharmaceutic , Nanoparticles/chemistryABSTRACT
SARS-CoV-2 is a particularly transmissible virus that causes a severe respiratory disease known as COVID-19. Safe and effective vaccines are urgently needed to combat the COVID-19 pandemic. The receptor-binding domain (RBD) of SARS-CoV-2 spike protein elicits most neutralizing antibodies during viral infection and is an ideal antigen for vaccine development. In particular, RBD expressed by E. coli is amenable to low cost and high-yield manufacturability. The adjuvant is necessitated to improve the immunogenicity of RBD. IC28, a TLR5-dependent adjuvant, is a peptide from bacterial flagellin. Mannan is a ligand of TLR-4 or TLR-2 and a polysaccharide adjuvant. Here, IC28 and mannan were both covalently conjugated with RBD from E. coli. The conjugate (RBD-IC28-M) elicited high RBD-specific IgG titers, and a neutralization antibody titer of 201.4. It induced high levels of Th1-type cytokines (IFN-γ) and Th2-type cytokines (IL-5 and IL-10), along with high antigenicity and no apparent toxicity to the organs. The mouse sera of the RBD-IC28-M group competitively interfered with the interaction of RBD and ACE2. Thus, conjugation with IC28 and mannan additively enhanced the humoral and cellular immunity. Our study was expected to provide the feasibility to develop an affordable, easily scalable, effective vaccine SARS-CoV-2 vaccine.
Subject(s)
COVID-19 , Viral Vaccines , Humans , Mice , Animals , COVID-19 Vaccines , SARS-CoV-2 , Mannans , Pandemics/prevention & control , Escherichia coli , COVID-19/prevention & control , Mice, Inbred BALB C , Antibodies, Neutralizing , Peptides , Cytokines , Antibodies, ViralABSTRACT
Zika virus (ZIKV) induces neurological and autoimmune complications such as microcephaly and Guillain-Barre syndrome. Effective vaccines are necessary to prevent the ZIKV infection. E protein of ZIKV is responsible for virus attachment, entry, and fusion. The domain III of E protein (EDIII) contains the neutralizing epitopes and is ideal to act as an antigen for ZIKV vaccine. However, EDIII is poorly immunogenic. CRM197 is a carrier protein and can activate T helper cells for EDIII. Mannan is a ligand of TLR-4 or TLR-2. Eight-arm PEG can link multiple EDIII molecules in one entity. In the present study, EDIII was covalently conjugated with CRM197, 8-arm PEG and mannan to improve the immunogenicity of EDIII. The conjugate (CRM-EDIII-PM) elicited high EDIII-specific antibody titers in the BALB/c mice. Th1-type cytokines (IFN-γ and IL-2) and Th2-type cytokines (IL-5 and IL-10) were secreted at a marked level. Thus, CRM-EDIII-PM could stimulate potent humoral and cellular immune response to EDIII. The serum exposure of CRM-EDIII-PM to the immune system was prolonged. Moreover, CRM-EDIII-PM did not lead to apparent toxicity to the organs. Therefore, CRM-EDIII-PM was expected as a promising vaccine candidate for its ability to induce strong immune responses.
Subject(s)
Mannans/chemistry , Polyethylene Glycols/chemistry , Viral Proteins/immunology , Viral Vaccines/immunology , Zika Virus/immunology , Animals , Antibodies, Viral/immunology , Antibody Affinity/immunology , Antibody Formation/immunology , Bacterial Proteins/toxicity , Chromatography, Gel , Circular Dichroism , Cytokines/metabolism , Dynamic Light Scattering , Electrophoresis, Polyacrylamide Gel , Female , Immunoglobulin G/metabolism , Mannans/toxicity , Mice, Inbred BALB C , Polyethylene Glycols/toxicity , Protein Domains , Rats, Sprague-Dawley , Spectrometry, Fluorescence , Spectroscopy, Fourier Transform Infrared , Toxicity Tests , Viral Proteins/chemistry , Viral Proteins/isolation & purification , Viral Proteins/pharmacokineticsABSTRACT
SARS-CoV-2 is responsible for a disruptive worldwide viral pandemic, and renders a severe respiratory disease known as COVID-19. Spike protein of SARS-CoV-2 mediates viral entry into host cells by binding ACE2 through the receptor-binding domain (RBD). RBD is an important target for development of virus inhibitors, neutralizing antibodies, and vaccines. RBD expressed in mammalian cells suffers from low expression yield and high cost. E. coli is a popular host for protein expression, which has the advantage of easy scalability with low cost. However, RBD expressed by E. coli (RBD-1) lacks the glycosylation, and its antigenic epitopes may not be sufficiently exposed. In the present study, RBD-1 was expressed by E. coli and purified by a Ni Sepharose Fast Flow column. RBD-1 was structurally characterized and compared with RBD expressed by the HEK293 cells (RBD-2). The secondary structure and tertiary structure of RBD-1 were largely maintained without glycosylation. In particular, the major ß-sheet content of RBD-1 was almost unaltered. RBD-1 could strongly bind ACE2 with a dissociation constant (KD) of 2.98 × 10-8 M. Thus, RBD-1 was expected to apply in the vaccine development, screening drugs and virus test kit.