Description
Rotaviruses (RVs) in the family Reoviridae are the most common causative agents of severe gastroenteritis in children under five years of age, leading to significant morbidity and mortality worldwide, particularly in developing nations (Cohen et al., 2022). The RV virion is composed of an approximately 80 nm in diameter triple-layered protein capsid that encapsulates 11 segments of double-stranded RNA genome. When observed under an electron microscope, rotavirus appears as a particle with short protrusions and a smooth outer edge, resembling a wheel shape (Figure 1). These segments encode six structural viral proteins (VP1 to VP4, VP6, and VP7) and five or six nonstructural proteins (NSP1 to NSP5/6). The RV capsid consists of an inner shell formed by VP2, an intermediate shell made by VP6, and an outer coat constituted by two surface proteins, VP4 and VP7. RVs are categorized into G and P genotypes based on the genes that encode VP7 and VP4. Known RVs are named according to their G and P type combinations, such as G1P8 and G2P4. Among the G types, G1, G4, and G9 are among the most prevalent in humans. On the other hand, P8 and P4 RVs are the two most frequent P types (Todd et al., 2010), accounting for the majority of detected RVs in humans globally; P6 is the third most prevalent P-type that often circulates in developing countries, especially in Africa, and accounts for up to 30% of the detected human RVs (HRVs) (Ouermi et al., 2017; Santos & Hoshino, 2005)
Figure 1. Diagram of the structure of Rotaviruses
Before the introduction of RV vaccines, most young children would be infected by RVs at least once before reaching the age of five (Crawford et al., 2017). After RV vaccines were implemented in 2006, RV-associated morbidity and mortality were substantially reduced (Burnett et al., 2017). However, unlike their high efficacy (80%-90%) in the industrialized world, the current live RV vaccines, including the most widely used Rotarix and RotaTeq vaccines, show significantly reduced effectiveness in low and middle-income countries (LMICs), with efficacy dropping to 40% to 60% (Clark et al., 2019; Soares-Weiser et al., 2019). As a result, despite the application of the present oral vaccines, RV infection still causes 130,000 deaths, 2.3 million hospitalizations, and 24 million outpatient visits among children under five years of age per annum, along with economic losses of over 1 billion US dollars each year (Parashar et al., 2006; Tate et al., 2012; Walker et al., 2013). Thus, RV-associated gastroenteritis continues to be a significant threat to global public health. Since most RV infections occur in LMICs, an improved RV vaccine strategy for better effectiveness and safety for these resource-deprived countries is in high demand.
While further study is needed to fully understand the causes of the reduced efficacy of the present live attenuated oral RV vaccines in LMICs (Desselberger, 2017; Parker et al., 2018), many studies have indicated that several intestine-related issues, such as coinfections with other enteric pathogens (Taniuchi et al., 2016), coadministration of the oral poliovirus vaccine (Parker et al., 2018; Ramani et al., 2016), malnutrition (Rytter et al., 2014), changes in microbiome composition (Harris et al., 2018), and different phenotypes of histo-blood group antigens (HBGAs) in the gut (Bucardo et al., 2019), impact the intestinal environment and thus decrease the replication and efficacy of the oral RV vaccines (Parker et al., 2018). Another potential issue associated with live RV vaccines is the increased risk of intussusception (Glass & Parashar, 2014; Rosillon et al., 2015; Yih et al., 2014), which likely results from the replication of the oral vaccines within the intestine.
To address the above concerns, nonreplicating subunit RV vaccines for parenteral administration have been proposed to bypass the intestine-related factors for enhanced efficacy and safety of RV vaccines, especially for use in LMICs (Pitzer et al., 2019). VP4 is an essential target for subunit RV vaccine development. VP4 proteins constitute the RV surface spikes that are explicitly digested by trypsin in the intestine into VP8* and VP5* fragments, a process that has been demonstrated to activate RVs for increased infectivity. Structurally, each VP4 protein consists of a distal head that is made up of VP8*, as well as a body/stalk region and a foot section that is constituted by VP5*. The VP5* foot section is embedded in the outer layer capsid that VP7 forms, while the VP8* head and the VP5* body/stalk region protrude outward, forming the surface spikes. Functionally, it is known that the VP8* head interacts with glycan receptors for RV attachment to host cells (Hu et al., 2012; Jiang et al., 2017; Tan & Jiang, 2014), whereas the VP5* body/stalk mediates RV cell membrane penetration to initiate RV infection (Arias & Lopez, 2021; Hu et al., 2012). Therefore, VP4 ectodomain, VPAe, consisting of both VP8* head and VPS* body/stalk, is a critical neutralizing antigen and an excellent vaccine target candidate. It was found that the truncated VP4 (aa26-476) containing VP8 and the stalk domain of VP5 could be expressed in soluble form in E. coli and purified to homogeneous trimers. The serum-neutralizing activity and protective efficacy of the VP4 (aa26-476) were significantly higher than that of VP8* and VP5* alone (Li et al., 2018). Representation of the domains of VP4. The atomic structure of VP4 (PDB entry 3IYU) is shown in Figure 2. The morphology of the VP4 protein in a mature virion is described as the head, body, stalk, and foot. The body and foot domains comprise the same peptides (aa248-476).
Figure 2. Schematic representation of rotavirus VP4 and the truncated VP4 proteins
Several proteins, such as the Foldon motif, have been artificially designed to possess trimeric structural features. Foldon is the C-terminal region of T4 phage fibrin, which spontaneously forms a typical trimer. The Foldon motif maintains a stable structure via non-covalent bonding, and the trimer retains high structural stability. It has been used as a trimerization motif coexpressed with HIV-1, gp140, SARS-CoV-2 RBD, and other trimeric proteins (Ahn, 2022; Ringe et al., 2015; Yang et al., 2002). However, although Foldon can effectively promote the target protein to form a trimer, its molecular weight is significant and has strong immunogenicity. Immunization with the fusion trimer antigen of Foldon and viral protein produced a robust immune response to Foldon protein and weakened the antigenic response to the viral protein. Therefore, other trimeric strategies need to be established for vaccine antigen design.
In recent years, synthetic biology technology has undergone rapid advancement, demonstrating significant application potential across various domains. By integrating genetic engineering, molecular biology, structural biology, and other methods, people can precisely design and synthesize proteins with specific structures and functions, such as recombinant humanized collagen. Collagen in the human body is rich in content and is involved in forming skin, bone, ligament, cornea, and other tissues. In the human body, there are at least 28 types of collagen. TypeⅢ collagen widely distributes in the skin, blood vessels, and other tissues. In this study, a collagen trimer was fused to the N-terminus of VP4 (aa26-476) to generate a collagen-VP4 fusion protein. This novel approach aims to develop a recombinant rotavirus vaccine with enhanced immunogenicity and immunoprotection.
VP4 (aa26-476) was proved to be a homogeneous trimer as expressed in E. coli. Different from the monomeric form, the trimeric proteins exhibit superior immunogenicity. Several trimerization motifs, such as Foldon derived from phage T4 fibritin, have been used to promote the formation of trimeric proteins with natural conformations. While Foldon-induced trimeric proteins display stability, their heightened immunogenicity poses limitations on their utility in vaccine antigen development. The human collagen-type protein is characterized by a triple-helix conformation. This study utilizes human collagen typeⅢ or artificially hydroxyproline-free ABC heterotrimeric collagen mimetic system to act as a trimerization motif (Jalan & Demeler, 2013; Rutschmann et al., 2014). The trimerization motif can be fused with VP4 protein through synthetic biology to form recombinant VP4 antigen.
i. Prokaryotes lack enzymes that hydroxylate prolyl4-hydroxylases (P4H), which is essential for the stability of collagen. In the future, P4H derived from giant viruses will be co-expressed with the Vp4 protein mentioned above to achieve the stability of Vp4 oligomers.
Figure 3. Schematic Diagram of P4Hs Mechanism
ii Further, we used the reported artificially designed protein-free heterotrimer-like collagen moieties ((PKG) n, (DKG) n, (EPG) n) to fusion three serological Vp4 proteins (P4, P8, P15) with one of the heterotrimer-like collagen moieties. The oligomers were formed by mixing the purified protein in vitro. In this way, a multitier-oligomer rotavirus vaccine can be prepared in Escherichia coli without P4H modification.
Figure 4. Schematic diagram of fusion between protein-free heterotrimer-like collagen motifs and antigenic VP4 proteins
A versatile collagen-fused VP4 trimer is being developed to engineer a virus-like particle (VLP) vaccine targeting human rotavirus, aiming to elicit a robust immune response. VLP vaccines have been utilized to combat human papillomavirus (HPV) and hepatitis B due to the non-infectious nature of virus-like particles, meaning that receiving the vaccine does not result in any symptoms of the virus. In this case, VP4 is used as an antigen, and human-derived collagen or collagen-like proteins are engineered to form a new trimer motif, which is fused with the VP4 protein to produce the trimer antigen. Additionally, proline hydroxylase P4H is co-expressed to ensure the stability of the collagen trimer. Additionally, the vaccine formulation involves the utilization of an artificially designed proline-free heterotrimer collagen motif.
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