Coding sequences of mZP2-3 (containing three copies of mZP2 peptide, TDVRYKDDMYHFFCPAIQA) and mIzumo1-3 (containing three copies of mIzumo1 peptide, LDCGERHIEVHRSEDLVLDCL) constructs ( Figure 1 ) were designed with optimized codons for expression in N. benthamiana. Gene constructs were synthesized by Eurofins Genomics GmbH (Ebersberg, Germany) and cloned into the pEX-A vector. Coding regions were flanked by BsaI restriction sites after polymerase chain reaction (PCR) amplification. PCR products were cloned separately into the pJET1.2 cloning vector (CloneJET PCR Cloning Kit; Thermo Fisher Scientific, Waltham, Massachusetts, USA). Coding sequences were cloned into a tobacco mosaic virus (TMV)-based expression vector of the MagnICON transient expression system, pICH31120, with BsaI restriction and ligation (Engler et al., 2008). MagnICON vectors were kindly provided by Nomad Bioscience GmbH (Halle/Saale, Germany).

Recombinant expression vectors were separately introduced into Agrobacterium tumefaciens strain ICF320 via electroporation. The resulting transformed colonies (confirmed using colony PCR and restriction enzyme digestion) were inoculated into 5 mL of Luria–Bertani (LB) medium supplemented with 50 μg/mL kanamycin and 50 μg/mL rifampicin and incubated overnight at 28°C with shaking (220 rpm). To prepare the main bacterial culture, 2 mL of the Agrobacterium suspension was placed in 200 mL LB medium containing the same antibiotics and incubated overnight (220 rpm, 28°C). Agrobacterium cells were harvested at 4560 × g for 30 min and resuspended in the infiltration medium (10 mM 2-(N-morpholino) ethanesulfonic acid [pH 5.8], 10 mM MgSO₄, and 0.02% v/v Silwet Gold) to achieve a final OD₆₀₀ of 0.15–0.2. Bacterial suspension was then vacuum-agroinfiltrated into the leaves of 6–8-week-old greenhouse-grown N. benthamiana plants as previously described (Nausch and Broer, 2017).

Agroinfiltrated N. benthamiana leaves were harvested at various days post-infiltration (dpi), frozen at −80 °C, lyophilized using a freeze dryer system (Alpha 1–4 LD; Martin Christ GmbH, Osterode, Germany), and pulverized using a mixer. Small-scale protein extraction was performed to evaluate the protein expression and accumulation. Total soluble protein (TSP) was extracted from 25 mg of lyophilized leaf material, homogenized in 500 µL cold protein extraction buffer (100 mM NaCl, 10 mM KCl, 6.5 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.2) plus complete protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany), using a Precellys 24 homogenizer (Bertin Instruments, France), and incubated for 30 min on ice. Clarified extract was obtained via repeated centrifugation at 15000 × g for 15 min at 4°C. Then, protein concentration of the supernatant was measured using the Pierce Coomassie Plus (Bradford) assay kit (Thermo Fisher Scientific) with bovine serum albumin (BSA) (Thermo Fisher Scientific) as the standard.

Histidine (His)-tagged recombinant mZP2-3 and mIzumo1-3 were partially purified via immobilized metal affinity chromatography (IMAC) using a Ni-chelating resin. Leaf extracts (in 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM sucrose, 5 mM imidazole) were centrifuged at 16,000 × g and 4°C for 1 h. Supernatants were collected and loaded onto a column (Bio-Rad Laboratories, Hercules, California, USA) containing pre-equilibrated Nuvia IMAC Ni-charged resin (Bio-Rad Laboratories). The column was washed twice with the wash buffer containing 20 and 30 mM imidazole. Recombinant protein was eluted with an elution buffer containing 300 mM imidazole, and the elution fraction was concentrated and desalted using Vivaspin 20 centrifugal concentrators with a 10 kDa cut-off membrane (Sartorius AG, Germany). LC-MS analyses of partially purified mIzumo1-3 protein were performed as already described (Klähn et al., 2021).

Protein samples were denatured at 95°C for 5 min in a loading buffer (10% glycerin, 150 mM Tris [pH 6.8], 3% SDS, 1% β-mercaptoethanol, and 2.5% bromophenol blue) and subjected to 12% SDS-PAGE. The separated proteins were transferred onto a 0.45-μm Hybond ECL nitrocellulose membrane (GE Healthcare Europe GmbH, Freiburg, Germany) using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad) at 100 V for 30 min. The membrane was blocked with Tris-buffered saline with Tween 20 (TBST; 20 mM Tris, 150 mM NaCl, and 0.05% [v/v] Tween20, pH 7.6) containing 5% (w/v) non-fat milk powder at 20–22°C (room temperature [RT]) for 2 h. After washing thrice with TBST, the membrane was probed with mouse monoclonal anti-His antibodies (Dianova, Hamburg, Germany) at a 1:1,000 dilution in Signal Boost ImmunoReaction Enhancer solution I (cat. No. 407207; Merck KgaA, Darmstadt, Germany) at RT for 2 h. After washing, the membrane was probed with horseradish peroxidase (HRP)-conjugated donkey anti-mouse antibodies (Dianova) at 1:10,000 dilution at RT for 1 h. After a final wash with TBS, signals were detected via incubation with ECL chemiluminescence reagent and subsequent exposure of the membrane to a Kodak Biomax light X-ray film (VWR; Darmstadt, Germany).

Animal experiments were approved by Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei (Mecklenburg-Vorpommern, Germany; approval No. 7221.3-1-071/20-2) and performed in accordance with the German animal protection regulations.

Two groups of female BALB/c mice (6–8-weeks-old, n = 10 per group) were used for the immunization study (BIOSERV Analytik; Rostock, Germany). Each animal in the treatment group was injected subcutaneously with a physical mixture of partially purified plant-produced mZP2-3 and mIzumo1-3, containing approximately 5 µg of mZP2 and mIzumo1 peptides, respectively. Control group received similarly prepared extracts from pICH31120-infiltrated leaves. Mice were immunized four times at three-week intervals. All doses contained 10% Polygen (MVP Lab) as an adjuvant. Serum samples were collected prior to primary immunization and 20 days after each immunization to measure the antibody levels. Female BALB/c mice were mated with proven fertile BALB/c males of a similar age four weeks after the final immunization. Males were removed after two weeks, and the females were allowed to litter. Fertility was defined as the mean number of pups born.

Specific IgG responses in sera were determined using ELISA. Partially purified mZP2-3 and mIzumo1-3 were used to coat a 96-well plate. After incubation at RT for 2 h, the plates were washed thrice with phosphate-buffered saline (PBS) containing 0.05% Tween 20 and blocked with PBS containing 1% (w/v) BSA for 1 h at RT. After washing with PBST, diluted sera (1:25,000 in PBS) were added to the wells and plates were incubated for 2 h at RT. After washing with PBST, the plates were incubated with HRP-conjugated donkey anti-mouse secondary antibodies (Dianova) at a dilution of 1:2000 in PBS at 37°C for 1 h. After washing, the wells were incubated with tetramethylbenzidine substrate for 10 min in the dark, reaction was stopped using 250 mM H₂SO₄, and the OD at 450 nm was measured using a plate reader (Bio Tek; Bad Friedrichshall, Germany).

On day 135 of the experiment, mice were sacrificed. To monitor the effect of immunization on follicular development, ovaries were collected, fixed in 4% paraformaldehyde, and embedded with paraffin. Ovaries were sectioned at 5 µm, stained with hematoxylin and eosin, and observed under a microscope.

Ovarian sections of immunized BALB/c and unimmunized wild mice were obtained from BIOSERV Analytik (Rostock, Germany). Ovarian sections were deparaffinized and subjected to antigen retrieval using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). Sections were blocked with 10% goat serum blocking solution (Life Technologies, Frederick, Maryland, USA) at RT for 1 h, washed with PBS, and incubated with 1X mouse-on-mouse IgG blocking solution (Invitrogen, Thermo Fisher Scientific) at RT for 1 h. Sections of unimmunized wild mice were incubated with a 1:20 dilution of serum samples from vaccinated BALB/c mice and kept at 4°C overnight. Sections of immunized BALB/c mice were incubated with a blocking solution (without the addition of any serum samples), washed, and incubated with 10 µg/mL fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibody (Invitrogen, Thermo Fisher Scientific) at 37°C for 1.5 h. After washing, slides were mounted with the DABCO mounting medium (25 mg/mL DABCO, 90% glycerol, and 10% PBS; pH 8.5) and observed under a fluorescence microscope.

Results are presented as the mean ± standard deviation. Experimental data were analyzed using IBM SPSS statistical software version 27. One-way analysis of variance and Student’s t-test were used to compare the means. Duncan’s test was used as a post-hoc test to measure the specific differences between the means. Statistical significance was set at p < 0.05.

To determine the transient expression of peptides mZP2 (TDVRYKDDMYHFFCPAIQA) and mIzumo1 (LDCGERHIEVHRSEDLVLDCL) in N. benthamiana, codon-optimized constructs were cloned separately into the expression vector, pICH31120, of the MagnICON system. Expression cassettes were introduced separately into N. benthamiana leaves via agroinfiltration. Constructs mZP2-3 and mIzumo1-3 contained three repeated copies of antigenic peptides ( Figure 1 ). A His-tag was added for the detection and purification of proteins.

Infiltrated leaves were harvested over time, and the expression levels of mZP2-3 and mIzumo1-3 were determined using western blotting. A strong protein band with an expected size of approximately 13 kDa for monomeric proteins confirmed the expression of mZP2-3. Weak signals were detected at approximately 26 and 39 kDa, representing the dimeric and trimeric forms of the protein, respectively. Production of mZP2-3 was initiated on 4 dpi, increased until 8 dpi, and then decreased. In contrast, the control sample (pICH31120-infiltrated leaf extract) exhibited no signal ( Figure 2A ).

Plants infiltrated with the mIzumo1-3.1 construct, in which the His-tag was located in the C-terminal region, exhibited no protein band in the western blot using antibodies against His tag ( Figure 2B ). Therefore, a second construct (mIzumo1-3.2), with a His-tag at the N-terminus, was designed to express mIzumo1 in plants. Western blotting analysis of crude protein extracts from leaves infiltrated with mIzumo1-3.2 revealed a band of approximately 25 kDa corresponding to the dimer form of mIzumo1-3 protein ( Figure 2C ); therefore, mIzumo1-3.2 was used for subsequent experiments. The highest accumulation of mIzumo1-3 was detected on 5–6 dpi.

Infiltrated leaves developed a necrotic phenotype that was more severe in leaves expressing mIzumo1-3 than in those expressing mZP2-3 ( Figure 2D ). Optimal harvesting time points for mZP2-3 and mIzumo1-3 were determined to be 8 and 6 dpi, respectively, when the protein accumulation was high. Subsequently, severe leaf necrosis and wilting were observed along with a decrease in protein levels.

After protein extraction, mIzumo1-3 showed low stability in the extract. The stability of mIzumo1-3 protein was increased after the addition of protease inhibitors ( Figure 3A ) and storage at –80°C ( Figure 3B ).

Recombinant mZP2-3 and mIzumo1-3 were partially purified from the crude plant extracts under non-denaturing conditions using Ni²⁺-charged column chromatography. Analysis of the partially purified mZP2-3 via western blotting revealed a dominant band of approximately 13 kDa and bands with molecular mass consistent with the monomeric and oligomeric forms of the antigenic construct, respectively ( Figure 4A ). In western blotting of the partially purified mIzumo1-3, a dominant band of approximately 25 kDa corresponding to the expected size of the dimer, and a band at the expected size of the tetramer form of mIzumo1-3 were detected, similar to those observed in the western blots of crude leaf extracts ( Figure 4B ). As the monomeric form of mIzumo1-3 was not detected via western blotting, a mass spectrometric analysis of the partially purified protein was performed, which confirmed the presence of mIzumo1-3 protein ( Supplementary Material, Figure S1 ). ELISA revealed that the concentration of partially purified mZP2 was 29.3 µg/g dry weight (DW) and that of mIzumo1 was approximately 4.5 µg/gDW.

To determine their immunogenicity, mZP2-3 and mIzumo1-3 were partially purified on a large scale and used in a prime-boost immunization assay. A group of 10 female BALB/c mice was immunized subcutaneously (four times at three-week intervals) with a mixture of equal amounts (5 µg) of plant-produced mZP2-3 and mIzumo1-3 antigens ( Figure 5A ). Mice in the negative control group were immunized with similarly prepared control plant extracts.

Humoral IgG antibody responses from individual mice were analyzed using ELISA at 1:25000 dilution of pre-immune and immune sera using plant-produced mZP2-3 or mIzumo1-3 as antigens. After primary immunization, no significant anti-mZP2-3 immune responses were detected in the sera of immunized mice compared to the pre-immune sera collected before primary immunization and that from the control group. However, second immunization induced an IgG antibody response that was significantly (p < 0.0001) higher than that in the primary dose and control group. Each subsequent immunization significantly increased the anti-mZP2-3 antibody response ( Figure 5B ).

Immunized mice exhibited a more potent anti-mIzumo1-3 antibody response than that observed at the pre-immune levels, even after primary vaccination with a physical mixture of recombinant proteins ( Figure 5C ). Immunization with the second and third doses significantly (p <0.0001) increased the antibody levels against mIzumo1-3. Antibody response continued to increase after the last immunization; however, it was not significant compared with that after the third dose ( Figure 5C ). Although some absorption was detected in the serum samples of the control group, it was significantly (p < 0.0001) lower than that in the serum samples of the antigen-vaccinated group ( Figures 5B, C ).

Immunized female mice were mated with proven fertile male mice three weeks after the final vaccination and allowed to produce litters to determine the effect of immunization on mouse fertility. Mice in the group treated with mZP2-3 and mIzumo1-3 showed a 39% reduction in average fertility compared to that in the control group. Average litter size was reduced from 8.4 ± 0.86 (mean ± standard error of the mean) in the control group to 5.1 ± 0.64 in treatment group immunized with mixture of plant-produced mZP2-3 and mIzumo1-3 antigens. Although the difference in mean litter size between the groups was not significant, the number of pups born correlated with the level of individual antibodies against both mZP2-3 and mIzumo1-3 proteins (p < 0.05; Figure 6 ). Moreover, negative correlation between litter size and total antibody levels (anti-mZP2-3 + anti-mIzumo1-3) in individual mice was significant (p < 0.01; Supplementary Material, Figure S2 ).

To determine whether immunization with recombinant antigens negatively impacts the ovary, mouse ovaries were isolated for observation. Immunization with plant-produced antigens caused no oophoritis or damage to the ovaries as they appeared normal. Additionally, histological analysis of ovarian sections from immunized mice did not reveal any abnormal follicular development or follicular degeneration. Figure 7 shows the normal follicles/oocytes at various stages of growth and development.

Immunofluorescence assay was used to determine the presence of induced antibodies bound to ZP in immunized mice. In ovaries collected from mice immunized with a physical mixture of plant-produced mZP2-3 and mIzumo1-3, immunofluorescence was observed from ZP on the surface of oocytes, indicating that anti-mZP2 antibodies bind to ZP in vivo. Ovarian sections obtained from mice in the control group failed to show any signals from ZP ( Figure 8A ).

Immune serum samples obtained after the final injection of plant-produced antigens were used for their reactivity with the ovarian sections of wild mice in an indirect immunofluorescence assay. When serum antibodies produced in vaccinated BALB/c mice were applied to the ovarian sections from unvaccinated wild mice, fluorescence signals were observed around the oocyte, indicating the binding of antibodies to native ZP. Notably, ovarian sections treated with pre-immune serum did not show any antibody reactions with the ZP matrix ( Figure 8B ). Reactivity of the immune sera with spermatozoa was not evaluated as acrosome-reacted mouse sperms could not be obtained for this study.