ZF2001, A Protein Subunit Vaccines against SARS-CoV-2

Abstract

Anhui Zhifei Longcom ‘s Zifivax, also known as ZF2001 (ZF-UZ-VAC-2001) is a protein subunit vaccine using a dimeric form of the receptor-binding domain (RBD) as the antigen, a harmless piece of the SARS-Cov-2 virus. As of June, 2022, over 300 million doses of Zifivax have been vaccinated with localized production in China base and Tashkent, Uzbekistan. At present, the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) is constantly mutating and evolving, and the coronavirus disease 2019 (COVID-19) epidemic is seriously threatening human health. Vaccination is the most effective and economical method to prevent and control the COVID-19 pandemic. Research institutions and companies around the world are employing various techniques to develop COVID-19 vaccines. According to the preparation technology, COVID-19 vaccines can be classified as inactivated virus vaccines, live attenuated vaccines, mRNA vaccines, DNA vaccines, viral vector vaccines, virus-like particle vaccines and protein subunit vaccines. Among these, viral protein subunit vaccines based on in vitro production of key viral proteins or peptides from bacterial, yeast, insect or mammalian cells have been drawing attention owing to their advantages of high safety and effectiveness, low cost of production, storage and transportation. Givrn this, this study reviewed the research and development status of ZF2001, as a reference for the development of protein subunit vaccines against SARS-Cov-2.

1. Introduction

The coronavirus disease 2019 (COVID-19) is an acute respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). As of April 8, 2022, COVID-19 has spread to 227 countries, causing 490 million infections and 6.17 million deaths [1]. The raging COVID-19 has seriously affected the global economy and public health. As the most effective and economical means to prevent and control COVID-19, COVID-19 vaccines have been attracting much attention, especially their R and D progress. According to the preparation technology, COVID-19 vaccines can be classified as inactivated virus vaccines, live attenuated vaccines, mRNA vaccines, DNA vaccines, viral vector vaccines, virus-like particle vaccines and protein subunit vaccines [2, 3]. Among these, protein subunit vaccines based on in vitro production of key viral proteins or peptides from bacterial, yeast, insect or mammalian cells has been drawing attention because they: (1) contain no viral genetic material and are thus safer than inactivated vaccines; (2) do not feed viruses and can be produced in a production workshop of lower biosafety level; (3) use transgenic technology to achieve high yield and high purity expression of antigens and facilitate large-scale production; and (4) are easy to store and transport [4]. As of April 9, 2022, a total of 36 COVID-19 vaccines have been formally approved by the government’s public health department, including 14 types of protein subunit vaccines [5]. In view of this, this research studies the research and development status of some marketed SARS-CoV-2 protein subunit vaccines based on domestic and foreign scientific literature and clinical data, and summarizes its research and development principles and clinical effects, to provide a reference for the research and development of SARS-CoV-2 protein subunit vaccine.

2. R and D principles of SARS-CoV-2 protein subunit vaccine

SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus that encodes 16 non-structural proteins, 9 accessory proteins, and 4 major structural proteins. Among them, the structural proteins are envelope protein (E), membrane protein (M), nucleocapsid protein (N) and spike protein (S) [6]. The S protein present in the viral envelope as a homologous trimer consists of two functional subunits, S1 and S2 (see Figure 1). The S1 subunit contains a receptor binding domain (RBD), which recognizes the receptor-angiotensin-converting enzyme 2 (ACE2) on host cells [7]. The S2 subunit contains fusion peptide (FP), junction region (CR), heptad repeat (HR), central helix (CH), etc., to fuse the membranes of viruses and host cells. When the S1 subunit binds to the ACE2 on host cells, the host protease recognizes and cleaves the S1/S2 cleavage site, the S1 subunit dissociates to fuse the membranes of FP that protrude after the conformational changes of the S2 subunit. Previous studies have shown that the RBD region of the S protein of SARS-CoV-2 is immunogenic and is the target of 90% neutralizing antibodies in immunosera [8]. Therefore, the S protein of SARS-CoV-2 has become the main target for the R and D of vaccines.

Figure 1.

Main targets for R and D of COVID-19 vaccine [6].

3. SARS-CoV-2 protein subunit vaccines produced based on RBD

RBD is one of the high-profile vaccine targets, while low immunogenicity limits its application in the vaccine. Adding antigen size, multimerization, or intensive antigen presentation in particles may enhance the immunogenicity of RBD subunit vaccine.

The research team of Gao Fu and Dai Lianpan from the Institute of Microbiology, Chinese Academy of Sciences, found in a study of RBD against MERS-coronavirus that disulfide-linked RBD dimers can induce higher neutralizing antibodies than traditional monomers. To further improve the stability and homogeneity of the dimeric antigen, the team optimized the dimeric protein structure and obtained tandem repeated RBD single-chain dimers. This tandem repeat single-stranded dimer has a single expression form, does not contain exogenous sequences, can maintain high vaccine potency, and is suitable for the R and D of COVID-19 and SARS-CoV-2 vaccines [9]. Based on this design strategy, Anhui Zhifei Longcom Biopharmaceutical Co., Ltd. and the Institute of Microbiology, Chinese Academy of Sciences jointly developed and produced the SARS-CoV-2 protein subunit vaccine ZF2001. In 2020, together with the Center of Advance Technology under the Ministry of Innovative Development of Uzbekistan, the multi-center international Phase 3 clinical trials were launched, data showed that the short-term and long-term protective efficacy against COVID-19 of any severity were 81.4% and 75.7%, respectively, that against Alpha variant was 92.7% and 88.3%, respectively, and that against Delta variant were 81.4% and 76.1%, respectively after three doses of ZF2001 in people over 18 years. This protective efficacy is higher than the WHO-preferred SARS-CoV-2 vaccine standard (70%) [10]. The sera of subjects vaccinated with three doses of inactivated vaccine or ZF2001 were tested by the pseudovirus cross-neutralization test, among which 62.5% sera of subjects vaccinated with inactivated vaccine were found to be positive for the Omicron variant neutralizing antibody while 100% sera of subjects vaccinated with ZF2001 (0, 1 and 5 months dose schedule) were found to be positive for Omicron variant neutralizing antibody [11]. By testing the neutralizing antibody titer, it was found that the titer of Omicron variant was reduced by 5.1-fold in the inactivated vaccine group and only 3-fold in the ZF2001 (0, 1 and 5 months dose schedule) group compared with the wild-type strain [11]. Sunney Xie’s team at Peking University showed that after two doses of inactivated vaccine, booster vaccination with ZF2001 induced a higher humoral immune response than with inactivated vaccine (CoronaVac) [12]. Based on the safety and efficacy of ZF2001, ZF2001 was approved for conditional marketing by the National Medical Products Administration on March 2, 2022, becoming the first domestic recombinant SARS-CoV-2 protein vaccine approved for conditional marketing, and will be used as a sequential vaccination to booster the protective effect of the existing vaccine.

4. Summary and outlook

Although it takes a long time to construct and screen engineered cell lines/strains with high expression of antigens in the early stage of the production of protein subunit vaccines, these vaccines have the advantages of high safety and immunogenicity, low production and transportation costs, which can meet the needs of low- and middle-income countries. In addition, COVID-19 protein subunit vaccines can be used as heterologous booster immunizations to trigger more ideal and long-lasting immune responses. Currently, multiple research teams and companies are actively developing COVID-19 protein subunit vaccines.

In order to develop a multivalent vaccine rapidly adapted to SARS-Cov-2 dominant variants, Gao Fu’s research team designed prototype-Beta and Delta-Omicron chimeric protein vaccines based on the WT homologous RBD dimer protein vaccine. Animal experiments have shown that the two-protein chimeric vaccine can stimulate a broader spectrum of antibody responses and protective effects. The Delta-Omicron chimeric protein vaccine provided better protection during the challenge test with Delta and Omicron variants [13]. At present, Zhifei Longcom has completed the construction of a cell bank for the Delta-Omicron chimeric protein vaccine.

Currently, billions of people worldwide have not been vaccinated with COVID-19 vaccines. Inequalities in vaccine access may lead to the emergence of more infectious variants. Therefore, it is necessary to continue the development of COVID-19 vaccines from multiple routes.