Currently two approaches can be used individually
Currently, two approaches can be used individually or combinedly to overcome weak immunogenicity of subunit 2-NBDG (Bookstaver et al., 2018; Moyle, 2017; Vartak and Sucheck, 2016). The first is to design particulate antigens. Virus-like particles (VLPs), for example—mainly derived from the capsid protein of pathogens—offer good immunogenicity owing to their relatively larger size (tens of nm) and repetitively arrayed epitopes. VLP-based vaccines are used against virus infection, such as from Hepatitis B virus, human papillomavirus, and Hepatitis E virus (Diaz-Arnold and Leary, 1989; Hanna and Bachmann, 2006; Li et al., 2005). The second approach, which is more often used for smaller antigens without the capacity for self-assembly into particles, incorporates potent immunostimulatory components called adjuvants, which are licensed for vaccine formulation, such as traditional aluminum salts, monophosphoryl lipid A (MPL), and squalene (Bonam et al., 2017; Shah et al., 2017). Subunit antigens can also be fused to carrier proteins, as they exhibit intra-molecular adjuvanticity and thereby increase the immunogenicity of the antigen; Diphtheria and Tetanus toxoids, meningococcal outer membrane protein complex (OMPC), and H. influenza protein D (HiD) have been used as carrier proteins in licensed vaccines (Pichichero, 2013).
Diphtheria toxin (DT) and its non-toxic mutant, CRM197 (Cross-Reacting Material 197) are well studied (Broker et al., 2011). DT consists of two fragments: fragment A (aa1–190), which contains the catalytic C domain, and fragment B, which consists of the transmembrane (T) and receptor-binding (R) domains. CRM197 harbors a mutation at position 52 on fragment A, which eliminates its cytotoxicity (Malito et al., 2012). However, CRM197 still retains strong immunogenicity and binds the DT-specific receptor, and therefore has been successfully employed in conjugated vaccines. Prevnar 13, for example, used for the prevention of pneumonia, is a conjugated vaccine composed of 13 serotypes of pneumococcal polysaccharide covalently conjugated to CRM197 (Gruber et al., 2012). Coupling to CRM197 (Dagan et al., 2010) aids in the rapid increase in Th1- and Th2-secreting T cells, and their subsequent production of cytokines, which, in turn, induces B cell proliferation and antigen-specific antibody secretion, thereby enhancing the immunogenicity of the polysaccharide to which it is conjugated. The benefit of CRM197 as an intra-molecular adjuvant has been shown with other proteins: Tobias and his group (Tobias et al., 2017) recently showed that a hybrid peptide located on human epidermal growth factor receptor 2 can induce an effective immune response and antitumor activity when grafted to CRM197. Thus, CRM197 has strong immunogenicity, non-toxicity, and can be potentially used as carrier protein.
Hepatitis E is a major public health issue, especially in developing countries, and is caused by infection with the Hepatitis E virus (HEV) (Aggarwal and Krawczynski, 2000; Sridhar et al., 2015). The HEV genome consists of three open reading frames (ORFs), of which the ORF2 encodes the virus capsid protein (Debing and Neyts, 2014). The truncated HEV capsid protein, E2 (aa394–606), naturally forms homodimers through hydrophobic interactions (Zhang et al., 2001) but is poorly immunogenic in mice and monkeys (Li et al., 2005). The p239 peptide from the ORF2 (aa368–606) is a recombinant vaccine against HEV derived from E2 through an N-terminal extension of 26 aa (Li et al., 2015; Zhang et al., 2015). Licensed in China in 2012, this vaccine is a potent particulate antigen. p239 and E2 share a common region (aa459–606) that encompasses numerous immune-dominant neutralizing epitopes and is responsible for virus-host interactions. However, comparably, the E2 protein has poor immunogenicity (Li et al., 2005).
Materials and methods
Discussion Previous studies have shown that CRM197 increases the production of Th1- and Th2-secreting T cells during the immune response and subsequently induces the differentiation and maturation of B cells by heterogeneous cytokines. Therefore, a stronger antibody response and immunological memory are elicited when the CRM197 carrier protein is combined with a capsular polysaccharide (Guttormsen et al., 1999; Kamboj et al., 2001). CRM197 has been used as a carrier protein in several licensed conjugated vaccines, including PREVNAR7, PREVNAR13 and HibTITER. In these vaccines, the polysaccharide is chemically covalently linked to the CRM197 protein. However, the immunogenic properties associated with combining CRM197 or the CRM197 functional domain with another protein via genetic fusion have not been completely evaluated. Fragment A, as the catalytic domain of DT, plays a pivotal role in cytotoxicity. Although the amino acid mutation at position 52 renders it enzymatically inactive, fragment A of CRM197 still retains the ability to bind the DT-specific receptor and stimulate a humoral and cellular immune response in the human body. Thus, we sought to determine whether the functional domain of CRM197 alone (i.e., fragment A) could function as a carrier protein.