A short mouse-specific peptide from zona pellucida 3 (mZP3, amino acids 328–342) has been shown to be associated with antibody-mediated contraception. In this study, we investigated the production of mZP3 in the plant, as an orally applicable host, and examined the immunogenicity of this small peptide in the BALB/c mouse model. The mZP3 peptide was inserted into the major immunodominant region of the hepatitis B core antigen and was produced in Nicotiana benthamiana plants via Agrobacterium-mediated transient expression. Soluble HBcAg-mZP3 accumulated at levels up to 2.63 mg/g leaf dry weight (LDW) containing ~172 µg/mg LDW mZP3 peptide. Sucrose gradient analysis and electron microscopy indicated the assembly of the HBcAg-mZP3 virus-like particles (VLPs) in the soluble protein fraction. Subcutaneously administered mZP3 peptide displayed on HBcAg VLPs was immunogenic in BALB/c mice at a relatively low dosage (5.5 µg mZP3 per dose) and led to the generation of mZP3-specific antibodies that bound to the native zona pellucida of wild mice. Oral delivery of dried leaves expressing HBcAg-mZP3 also elicited mZP3-specific serum IgG and mucosal IgA that cross-reacted with the zona pellucida of wild mice. According to these results, it is worthwhile to investigate the efficiency of plants producing HBcAg-mZP3 VLPs as immunogenic edible baits in reducing the fertility of wild mice through inducing antibodies that cross-react to the zona pellucida.

/pdf/oral-and-subcutaneous-immunization-with-a-plant-produced-imlgqr3g.pdf

Oral and Subcutaneous Immunization with a Plant-Produced Mouse-Specific Zona Pellucida 3 Peptide Presented on Hepatitis B Core Antigen Virus-like Particles

Rodent population control through contraception requires species-specific oral contraceptive vaccines. Therefore, in this study, we produced putative mouse-specific contraceptive peptides, mZP2 (from oocyte) and mIzumo1 (from sperm), in plants using Agrobacterium-mediated transient expression. Peptides were produced separately in Nicotiana benthamiana using constructs encoding antigens containing three copies of each peptide. We also determined the immunogenicity and contraceptive effects of the plant-produced antigens in female BALB/c mice. Mice immunized subcutaneously with a relatively low amount of antigen (5 µg/dose of each peptide in a mixture) showed systemic immune responses against mZP2-3 and mIzumo1-3 antigens. Moreover, the mean litter size of mice treated with the plant-produced antigens was reduced by 39% compared to that of the control mice. Notably, there was a significant negative correlation between the number of pups born and individual antibody levels against both antigens. Immunofluorescence assays demonstrated the binding of induced antibodies to the oocytes of BALB/c and wild-type mice in vivo and in vitro, respectively. Our study demonstrate the feasibility of producing small contraceptive peptides in plants that can be further used to develop oral contraceptive vaccines against mouse populations.

/pdf/immunogenicity-and-contraceptive-efficacy-of-plant-produced-2yg78djf.pdf

Immunogenicity and contraceptive efficacy of plant-produced putative mouse-specific contraceptive peptides

Nonenveloped virus-like particles (VLPs) are self-assembled oligomeric structures composed of one or more proteins that originate from diverse viruses. Because these VLPs have similar antigenicity to the parental virus, they are successfully used as vaccines against cognate virus infection. Furthermore, after foreign antigenic sequences are inserted in their protein components (chimVLPs), some VLPs are also amenable to producing vaccines against pathogens other than the virus it originates from (these VLPs are named platform or epitope carrier). Designing chimVLP vaccines is challenging because the immunogenic response must be oriented against a given antigen without altering stimulant properties inherent to the VLP. An important step in this process is choosing the location of the sequence modifications because this must be performed without compromising the assembly and stability of the original VLP. Currently, many immunogenic data and computational tools can help guide the design of chimVLPs, thus reducing experimental costs and work. In this study, we analyze the structure of a novel VLP that originate from an insect virus and describe the putative regions of its three structural proteins amenable to insertion. For this purpose, we employed molecular dynamics (MD) simulations to assess chimVLP stability by comparing mutated and wild-type (WT) VLP protein trajectories. We applied this procedure to design a chimVLP that can serve as a prophylactic vaccine against the SARS-CoV-2 virus. The methodology described in this work is generally applicable for VLP-based vaccine development. 

Computational method for designing vaccines applied to virus-like particles (VLPs) as epitope carriers.

Because of the time-consuming nature of surgical neutering and the rapid rate of reproduction among domestic cats, it is crucial to investigate alternative, nonsurgical methods of contraception for this species. Sperm protein IZUMO1 and its oocyte receptor JUNO have been proposed as potential targets for nonsurgical contraceptives. This study aimed to demonstrate (1) the protein coding sequence of feline IZUMO1 and JUNO, (2) gene expression in specific organs by measuring mRNA levels in different visceral tissues, and (3) the expression of IZUMO1 and JUNO during sperm maturation and folliculogenesis, respectively. Amplification for sequencing of feline IZUMO1 and JUNO was performed using the RT-PCR method. Levels of gene expression in different tissues were evaluated using real-time PCR. In situ hybridization was performed to localize JUNO mRNA in ovarian tissues. The complete coding sequences of IZUMO1 and JUNO were obtained and analyzed. A comparison between protein orthologs demonstrated the conservation of IZUMO1 and JUNO in Felidae. The real-time PCR results from various visceral organs indicated that IZUMO1 was significantly higher in the testis than in other organs, whereas JUNO was significantly higher in the ovary than in other organs. Expression of IZUMO1 was found to be higher in the testes than in the caput, corpus, and cauda of epididymides. In situ hybridization revealed that JUNO mRNA was in the ooplasm and nucleus of the primordial, primary, secondary, and antral follicles. Importantly, this was the first study to demonstrate the IZUMO1 and JUNO genes in the testis and ovary of cats. The results are useful for future research related to these genes and for developing contraceptives against these targets. 

Expression of IZUMO1 and JUNO in the gonads of domestic cats (Felis catus).

Here, we report that four functional fragments of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) spike protein including receptor binding motif (RBM), fusion peptide (FP), heptad repeat 1 (HR1) and heptad repeat 2 (HR2) were chosen to develop a recombinant S subunit protein vaccine. This recombinant protein consisting of S230 amino acids (aa) (S230) bound specifically to the antibody from COVID-19-patients serum, which showed very strong antigenicity. The S230 was then engineered to present on the surface of Hepatitis B core (HBc) virus-like particles (VLPs) to develop HBc-S230 chimeric VLPs vaccine. Both vaccines induced strong humoral and cellular immune responses in mice, however, HBc-S230 chimeric VLPs elicited significantly higher immunogenicity than the S230. HBc-S230 chimeric VLPs promoted to generate not only dramatically higher levels of S230-specific serum antibodies, but also marked higher CD4+/CD8 + T cells ratio and substantially higher yields of IFN-γ and IL-6. Furthermore, HBc-S230 chimeric VLPs induced serum antibodies that could effectively neutralize the infection with three SARS-CoV-2 pseudoviruses (Wild type, Delta and Omicron). Our results demonstrated that HBc-S230 chimeric VLPs immunization conveyed the humoral immunity, which lasted longer than six months. Clearly, HBc-S230 chimeric VLPs enhanced immunogenicity of the S230, which could provide potent and durable protection against SARS-CoV-2 infection, indicating that HBc-S230 chimeric VLPs possessed great potential for developing highly immunogenic vaccines against SARS-CoV-2. 

A novel HBc-S230 protein chimeric VLPs induced robust immune responses against SARS-CoV-2

Contraceptive vaccines are designed to stimulate autoimmune responses to molecules involved in the reproductive process. A mouse-specific peptide from zona pellucida 3 (mZP3) has been proposed as a target epitope. Here, we employed a plant expression system for the production of glycosylated mZP3 and evaluated the immunogenicity of plant-produced mZP3-based antigens in a female BALB/c mouse model. In the mZP3-1 antigen, mZP3 fused with a T-cell epitope of tetanus toxoid, a histidine tag, and a SEKDEL sequence. A fusion antigen (GFP-mZP3-1) and a polypeptide antigen containing three repeats of mZP3 (mZP3-3) were also examined. Glycosylation of mZP3 should be achieved by targeting proteins to the endoplasmic reticulum. Agrobacterium-mediated transient expression of antigens resulted in successful production of mZP3 in Nicotiana benthamiana. Compared with mZP3-1, GFP-mZP3-1 and mZP3-3 increased the production of the mZP3 peptide by more than 20 and 25 times, respectively. The glycosylation of the proteins was indicated by their size and their binding to a carbohydrate-binding protein. Both plant-produced GFP-mZP3-1 and mZP3-3 antigens were immunogenic in mice; however, mZP3-3 generated significantly higher levels of serum antibodies against mZP3. Induced antibodies recognized native zona pellucida of wild mouse, and specific binding of antibodies to the oocytes was observed in immunohistochemical studies. Therefore, these preliminary results indicated that the plants can be an efficient system for the production of immunogenic mZP3 peptide, which may affect the fertility of wild mice.

/pdf/plant-produced-mouse-specific-zona-pellucida-3-peptide-3hbxadfb.pdf

Plant-Produced Mouse-Specific Zona Pellucida 3 Peptide Induces Immune Responses in Mice

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Oral and Subcutaneous Immunization with a Plant-Produced Mouse-Specific Zona Pellucida 3 Peptide Presented on Hepatitis B Core Antigen Virus-like Particles

Plant-Produced Mouse-Specific Zona Pellucida 3 Peptide Induces Immune Responses in Mice

Immunogenicity and contraceptive efficacy of plant-produced putative mouse-specific contraceptive peptides