In the face of an overpopulation of mice, developing a species-specific, inexpensive, effective, and readily applicable contraceptive vaccine is required to allow oral application. This study aimed to develop an immunocontraceptive vaccine based on the mZP3 glycopeptide in plants.
To develop contraceptive vaccines targeting wild mouse populations, an effective mouse-specific antigen is needed. Vaccines based on the mZP3 peptide appear to be promising since the peptide has been found to be sufficiently immunogenic and contraceptive in wild mice (Hardy et al. 2002a). In addition, it is hypothesized to be mouse-specific because it has not been detected in other species such as hamster, guinea pig, cat or dog (East et al. 1985; Millar et al. 1989). Therefore, it might be a good choice for producing a mouse-specific contraceptive vaccine in transgenic plants that is a suitable platform for the rapid, low-cost and large-scale production of vaccines (Ma et al. 2003; Streatfield and Howard 2003; Sabalza et al. 2014). More importantly, transgenic plants can facilitate the administration of vaccines by providing a potential platform for the oral delivery of vaccines (Daniell et al. 2009; Chan and Daniell 2015), which could be an ideal method for vaccination of wild life populations.
To create an effective vaccine, it is necessary to produce a sufficient amount of stable antigen. Our results indicated that mZP3-1 was unstable, with accumulation levels dropping four days after infiltration, before the optimal day proposed for the expression system (Marillonnet 2004; Chen et al. 2013). The low mZP3-1 yield might be related to its susceptibility to proteolytic processes in the heterologous environment (Benchabane et al. 2008; Faye et al. 2005; Manning et al. 2010). Fusing recombinant peptides or proteins with a partner that is stably expressed in plants is one approach to increase the yield of target proteins in plants (Streatfield 2007). Fusion of mZP3-1 to the GFP coding region, resulting in GFP-mZP3-1, led to a more than 25-fold higher accumulation of mZP3. GFP has already been used successfully to improve protein production and stability under various conditions (Hsieh et al. 2010; Janczak et al. 2015; Ponndorf et al. 2017). This stabilization seems to depend not only on the GFP sequence but also on the protein size since mZP3-3 showed a similar effect on the stability and accumulation of the protein.
The low amounts of mZP3-1 could also be due to the massive damage that mZP3-1 production caused in the plants. It has already been observed that transient expression of ER-targeted proteins can put stress on plants and cause prohibitive levels of tissue necrosis that consequently lead to low yields of recombinant proteins (Diamos and Mason 2018; Hamorsky et al. 2015; Hussain et al. 2014). Expression of GFP-mZP3-1 and mZP3-3 reduced the severity of tissue necrosis and resulted in higher protein accumulation. Other studies have achieved an increase in target protein accumulation by reducing the ER stress response and moderating plant tissue necrosis (Diamos and Mason 2018; Margolin et al. 2020). Hence, differences in protein accumulation might be due not only to differences in protein stability but also to differences in the induction of plant stress responses.
The other necessity for an efficient vaccine is sufficient immunogenicity. Immunization of mice with mZP3-3 antigens led to the generation of significantly higher antibody responses against mZP3 compared to GFP-mZP3-1. In line with this, previous studies have shown that antigens containing multiple repeats of ZP3 epitopes stimulate higher antibodies in BALB/c mice compared with those containing single mZP3 epitopes (Sadler et al. 2000; Hardy et al. 2004). Incorporation of multiple copies of epitopes into antigens has been investigated previously for various weak immunogens to enhance their immunogenicity (Jinshu et al. 2004; Haigh et al. 2007; Sandam et al. 2021).
Notably, the production of mZP3 antigens in plants seems to increase immunogenicity compared to other production systems. E. coli-produced single mZP3-peptide antigens elicited no significant antibody responses to ZP3 in BALB/c immunized mice (Hardy et al. 2004), and ZP3 protein expressed in the insect cell-expression system (Hardy 2003) seems to induce lower levels of antibodies against mZP3 peptide compared to our study. These observations highlight the importance of the expression system for the production of recombinant ZP3 antigens and show the superiority of plants over other expression platforms in this case. Hardy 2003 demonstrated the importance of glycosylation in the efficiency of recombinant mouse ZP3 antigens as immunocontraceptive. Plants offer the ability to perform posttranslational modification of proteins in a similar, but not equal, way compared to mammalian proteins that can support the immunogenicity of plant-made vaccines (Streatfield and Howard 2003; Faye et al. 2005; Bosch and Schots 2010). Although glycosylation analysis was not carried out, the strong reaction of plant-produced proteins with the carbohydrate-binding protein Con A and the size of the proteins may be evidence for the glycosylation of the mZP3 peptide. Therefore, oligosaccharide residues present in plant-produced mZP3 may be a contributing factor in increasing the immunogenicity of antigens in vaccinated mice. Nevertheless, it might also indicate the stimulating effects of the TT epitope as an adjuvant. It has been observed previously that the antibody responses to ZP3 improved when additional appropriate T-cell helper epitopes were included in antigens (Jackson 1998; Sadler et al. 2000; Shrestha et al. 2014).
Wild mice cause huge problems in immunization and mating studies due to their aggressive behavior. Hence, we had to restrict studies with live animals to the laboratory BALB/c mice used in most previous studies, although they have not shown a significant reduction in fertility dependent on the induction of anti-mZP3 antibodies (Fitchen 1995; Hardy et al. 2002a; Hardy et al. 2004). Fertility of BALB/c mice was reduced by immunization with repeated mouse ZP3 peptides conjugated to multiple contraceptive epitopes and defined T helper cell epitopes (Sadler et al. 2000; Hardy et al. 2008). Accordingly, despite high anti-mZP3 antibody titers, reproduction rates were not significantly reduced in immunized mice. It has been suggested that contraception depends on additional factors such as mouse genetic background in combination with antibody levels (Fitchen 1995; Hardy et al. 2004). Immunization with mZP3 and other closely related peptides reduces fertility in some, but not all, mouse strains, despite inducing antibody responses. The ability to elicit a contraceptive response and the level of contraception induced by mouse ZP3 peptides varies depending on the genotype and strain of the mouse (East et al. 1984; Fitchen 1995; Tung et al. 1996; Hardy et al. 2002a; Hardy et al. 2004).
Nevertheless, as observed by (Hardy et al. 2002), immunofluorescence studies showed that serum anti-mZP3 antibodies could specifically react to the native ZP protein in the ovaries of wild mouse. The mZP3 antisera showed no binding to ZP in BALB/c mice, although sequence analysis revealed that mZP3 sequence is conserved between the two strains. It has been reported that under identical conditions, antibodies against mZP3 successfully reduced fertility in wild mice but had no effect on the fertility of BALB/c mice (Hardy et al. 2002). Hence, the immunological cross-reactivity between serum antibodies and ZP could indicate the ability of anti-mZP3 antibodies to bind to ZP in vivo in wild mice, which might lead to reduced fertility. Therefore, plant-produced mZP3 antigens can potentially be developed as contraceptive vaccines to manage wild mouse populations. Further research is needed to evaluate the contraceptive effect of plant-made mZP3 in wild mice.
In conclusion, the present study demonstrates that mZP3 can be effectively produced in N. benthamiana plants using a transient expression system. Compared to other production systems, plants seem to be more promising for producing highly immunogenic mZP3 antigens. Among the antigens studied, mZP3-3 is the best choice because it was efficiently expressed in the plant and induced high levels of antibodies that can react to the native zona pellucida of wild mice as the target species. Therefore, plant-produced mZP3-3 has the potential to be developed as an immunocontraceptive vaccine to control the populations of wild mice. Further research will be conducted to determine the contraceptive efficiency of plant-made mZP3-3 antigen in wild mice.