Project Manager
Tabynov Kaisar Kazybaevich
candidate of veterinary sciences, professor
Scopus Author ID: 55489111500
Researcher ID: J-4155-2015
ORCID: https://orcid.org/0000-0001-5823-1280
About the project
Relevance
As a new intranasal SARS-CoV-2 vaccine platform, the project research group proposes to develop and test a design based on chitosan nanoparticles with mannose. The complex of these natural biocompatible polymers, in addition to being an ideal transport vehicle of protein subunits into mucosal membranes, also have a pronounced adjuvant effect. This nanovaccine formulation was obtained by our partners from Ohio State University (USO, USA) and was previously used in the development of swine influenza and avian salmonellosis vaccines. To enhance the T-cell immune response of the nanovaccine formulation, we plan to include in its composition the adjuvant CpG55.2. (Toll-like receptor 9 antagonist), developed on the basis of artificial intelligence by our other partner Vaxine Pty Ltd (Adelaide, Australia). This adjuvant has its own unique sequence and is used as an important component of a subunit SARS-CoV-2 vaccine under the trade name COVAX-19 (developed by Vaxine Pty Ltd), which has successfully completed a Phase I clinical trial in 40 volunteers in Australia and is now being prepared for Phase II-III clinical trials in 10 countries worldwide. Importantly, this adjuvant has the potential to reduce the antigenic load of the vaccine by more than 400-fold.
The main idea of the proposed project is to combine two advanced nanoparticle-based intranasal antigen delivery technologies and the adjuvant CpG55.2. (TLR-9 antagonist) to create a new platform for the preparation of a safe, effective and non-injectable SARS-CoV-2 vaccine. Achieving the latter eliminates a number of problems (pain, cross-contamination, needle stick injury, under- or over-dosing, increased costs, and fear of injections) associated with vaccination.
Objective
The aim of the project is to develop a novel safe, immunogenic and effective SARS-CoV-2 nanovaccine platform with an intranasal route of administration.
Expected and achieved results:
1) Design and development of recombinant S1/RBD proteins of SARS-CoV-2 virus;
2) Development of a laboratory animal model to evaluate vaccine efficacy;
3) Obtaining a safe and effective formulation of SARS-CoV-2 nanovaccine for intranasal administration;
4) Evaluation of stability of SARS-CoV-2 nanovaccine formulation under storage and transportation conditions;
5) Study of immunobiologic properties of SARS-CoV-2 vaccine on laboratory animal model.
Plasmids (pCMV3-2019-nCoV-RBD) with genes encoding fragments of RBD Spike protein of SARS-CoV-2 virus were obtained. This plasmid was then recloned into the pFASTBac 1 vector. Next, bacmid DNA was produced using the Bac-to-Bac™️ Baculovirus Expression System (Gibco). The resulting baculovirus was subjected to full-genome NGS sequencing. Expression of RBD protein was performed in the Baculovirus Expression System (ExpiSf™ commercial kit) according to the manufacturer's protocol (Gibco). The resulting protein was purified by affinity chromatography on a Ni-NTA column and gel filtration on chromatography columns. The presence of protein in the purified material was detected by gel electrophoresis (SDS-PAGE) and Western blotting. Studies showed the obtained recombinant SARS-CoV-2 (2019-nCoV) Spike Protein (RBD) protein consists of 271 amino acids and has an approximate molecular mass of 30.3 kDa. The purity of the protein was more than 90% and the concentration was more than 10 mg.
A laboratory model of SARS-CoV-2 infection and its transmission in Syrian hamsters was experimentally characterized, which can be used to evaluate the efficacy of the COVID-19 vaccine under development.
A subunit vaccine based on recombinant monomeric RBD protein loaded in mannose-conjugated chitosan nanoparticles (NARUVAX-C19/Nano) was found to, provides the formation of antigen-specific IgA antibodies and Th1-cell immune responses in mice during double intranasal immunization at 21-day intervals, as well as significant protection against wild-type SARS-CoV-2 (D614G) virus infection in the Syrian hamster model.
It was found that the recommended optimal temperature for long-term storage and transportation (at least 12 months) of NARUVAX-C19/Nano vaccine is -20°C or +4°C, and short-term transportation of the preparation can be carried out at +22°C, but not more than 7 days.
Duration of postvaccinal protective immunity of NARUVAX-C19/Nano vaccine on hamster model in the mode of intranasal double immunization is 6 months.
It was found that NARUVAX-C19/Nano vaccine in the mode of double intranasal application is immunogenic in both newborn and old mice. The level of formation of serum anti-RBD IgA antibodies in vaccinated animals was significantly higher than that of the control group. It should be noted that the level of IgA antibodies in newborn mice, although not significantly, was higher than in old mice.
Plasmids (pCMV3-2019-nCoV-RBD) with genes encoding fragments of RBD Spike protein of SARS-CoV-2 virus were obtained. This plasmid was then recloned into the pFASTBac 1 vector. Next, bacmid DNA was produced using the Bac-to-Bac™️ Baculovirus Expression System (Gibco). The resulting baculovirus was subjected to full-genome NGS sequencing. Expression of RBD protein was performed in the Baculovirus Expression System (ExpiSf™ commercial kit) according to the manufacturer's protocol (Gibco). The resulting protein was purified by affinity chromatography on a Ni-NTA column and gel filtration on chromatography columns. The presence of protein in the purified material was detected by gel electrophoresis (SDS-PAGE) and Western blotting. Studies showed the obtained recombinant SARS-CoV-2 (2019-nCoV) Spike Protein (RBD) protein consists of 271 amino acids and has an approximate molecular mass of 30.3 kDa. The protein purity was greater than 90% and the concentration was greater than 10 mg. A laboratory model of SARS-CoV-2 infection and its transmission in Syrian hamsters has been experimentally validated and can be used to evaluate the efficacy of the COVID-19 vaccine under development.
A subunit vaccine based on recombinant monomeric RBD protein loaded in mannose-conjugated chitosan nanoparticles (NARUVAX-C19/Nano) has been found to produce antigen-specific IgA antibodies and Th1-cell immune responses in mice when immunized twice intranasally at 21-day intervals, provides the formation of antigen-specific IgA antibodies and Th1-cell immune responses in mice upon double intranasal immunization at 21-day intervals, as well as significant protection against wild-type SARS-CoV-2 (D614G) virus infection in the Syrian hamster model.
It was found that the recommended optimal temperature for long-term storage and transportation (at least 12 months) of NARUVAX-C19/Nano vaccine is -20°C or +4°C, and short-term transportation of the preparation can be carried out at +22°C, but not more than 7 days.
The duration of postvaccinal protective immunity of NARUVAX-C19/Nano vaccine on hamster model in the mode of intranasal double immunization is 6 months.
It was found that the NARUVAX-C19/Nano vaccine in the mode of double intranasal administration is immunogenic in both newborn and old mice. The level of formation of serum anti-RBD IgA antibodies in vaccinated animals was significantly higher than that of the control group. It should be noted that the level of IgA antibodies in newborn mice, although not significantly, was higher than in old mice.
Preclinical study (additional work under the project) of safety of the developed vaccine NARUVAX-C19/Nano on the basis of GLP testing laboratory of the National Center for Expertise of Medicines and Medical Devices of the Ministry of Health of the Republic of Kazakhstan (regulatory body). It is shown that the vaccine NARUVAX-C19/Nano has no toxic effect on the body, has good tolerability and does not show allergic effect in tests on laboratory animals, including pregnant individuals.
List of publications and patents
Based on the results of the conducted research, 3 (three) articles were published in peer-reviewed international scientific publications indexed by Web of Science (WoS) and Scopus (NPJ Vaccines, IF-9. 2, WoS-Q1, Scopus-95%; Human Vaccines & Immunotherapeutics, IF-4.8, WoS-Q1, Scopus-68%; Scientific Reports, IF-4.6, WoS-Q2, Scopus-92%), 2 (two) national invention patents were obtained and 1 (one) International patent application was filed:
1. Tabynov K, Turebekov N, Babayeva M, Fomin G, Yerubayev T, Yespolov T, Li L, Renukaradhya GJ, Petrovsky N, Tabynov K. An adjuvanted subunit SARS-CoV-2 spike protein vaccine provides protection against Covid-19 infection and transmission. NPJ Vaccines. 2022 Feb 23;7(1):24. doi: 10.1038/s41541-022-00450-8.
https://www.nature.com/articles/s41541-022-00450-8
2. Tabynov K, Solomadin M, Turebekov N, Babayeva M, Fomin G, Yadagiri G, Renu S, Yerubayev T, Petrovsky N, Renukaradhya GJ, Tabynov K. An intranasal vaccine comprising SARS-CoV-2 spike receptor-binding domain protein entrapped in mannose-conjugated chitosan nanoparticle provides protection in hamsters. Sci Rep. 2023 Jul 26;13(1):12115. doi: 10.1038/s41598-023-39402-0.
https://www.nature.com/articles/s41598-023-39402-0
3. Solomadin M, Tabynov K, Petrovsky N, Tabynov K. Evaluation of a SARS-CoV-2 spike protein ectodomain subunit vaccine with a squalene emulsion adjuvant in rodents and rhesus macaques. Hum Vaccin Immunother. 2023 Aug;19(2):2258571. doi: 10.1080/21645515.2023.2258571.
https://www.tandfonline.com/doi/full/10.1080/21645515.2023.2258571
4. Штамм hCoV-19/Kazakhstan/KazNAU-NSCEDI-481/2020 KKZI KA-294 вируса SARS-CoV-2, порядка Nidovirales семейства Coronaviridae рода Betacoronavirus вида Coronavirus клайда G, предназначенный для приготовления и тестирования иммунобиологических препаратов и определения противовирусной активности различных субстанций in vitro и in vivo. Патент на изобретение №34974 от 26 марта 2021 г.
https://gosreestr.kazpatent.kz/Invention/DownLoadFilePdf?patentId=337209&lang=ru
5. Ассоциация штаммов hCoV-19/Kazakhstan/KazNAU-NSCEDI-4635/2020 (Wuhan variant), hCoV-19/Kazakhstan/KazNAU-NSCEDI-Kaissar/2021 (Alpha variant), hCoV-19/Kazakhstan/KazNARU-NSCEDI-5526/2021 (Delta variant) вируса SARSCoV-2, предназначенная для приготовления и тестирования средств специфической профилактики, терапии, диагностики и дезинфекции в условиях in vitro и in vivo. Патент на изобретение № 35884 от 07 октября 2022 г.
https://gosreestr.kazpatent.kz/Invention/DownLoadFilePdf?patentId=359042&lang=ru
6. Международная заявка на патент WO/2023/159082 от 24.08.2023 «Nanotechnology based intranasal COVID-19 vaccine comprising chitosan», Inventors: GOURAPURA, Renukaradhya J.; YADAGIRI, Ganesh; KENNEY, Scott; SCHROCK, Jennifer; BOLEY, Patricia; TABYNOV, Kaissar; TABYNOV, Kairat; YESPOLOV, Tlektes. Applicants: Ohio State Innovation Foundation (US), Kazakh National Agrarian Research University (KZ). Опубликована в Derwent Innovations Index (WoS) под названием «Composition used to elicit or enhance an immune response to severe acute respiratory syndrome coronavirus 2, comprises severe acute respiratory syndrome coronavirus 2 antigen associated with a nanoparticle».
https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2023159082&_gid=202334
Information for potential users
If successful, the platform could be invested in and then licensed to local and possibly global pharmaceutical companies (foreign partners will support this). It could also serve as a basis for the development of vaccines against other respiratory viral infections, including those with pandemic potential.
Members of the research group
Tabynov Kairat
PhD, Associate Professor
Scopus Author ID: 57196167800
Researcher ID: N-8761-2017
ORCID: https://orcid.org/0000-0001-9411-7952
Turebekov N.A.
PhD
Scopus Author ID: 57195575537
Researcher ID: AAZ-2754-2020
ORCID: https://orcid.org/0000-0001-6826-2611
Belousov V.Yu.
Candidate of Biological Sciences.
Scopus Author ID: 58030218400
Researcher ID: HJS-1618-2023
Solomandin M.V.
Scopus Author ID: 57996655100
Researcher ID: IAE-8952-2023
Orynbasar M.B.
Scopus Author ID: 57563473400
Researcher ID: FPZ-6475-2022
Yelchibaeva Leyla
Master of Veterinary Science
Scopus Author ID: 57563224400
Researcher ID: EGQ-1571-2022
ORCID: https://orcid.org/0000-0001-6895-7720