The evaluation of novel oral vaccines based on self-amplifying RNA lipid nanparticles (saRNA LNPs), saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum to neutralize SARS-CoV-2 variants alpha and delta

The aim of this study was to present and evaluate novel oral vaccines, based on self-amplifying RNA lipid nanparticles (saRNA LNPs), saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum, to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) variants alpha and delta. After invitro evaluation of the oral vaccines on HEK293T/17 cells, we found that saRNA LNPs, saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum could express S-protein at both mRNA and protein levels. In the next step, BALB/c mice were orally vaccinated with saRNA LNPs, saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum at weeks 1 and 3. Importantly, a high titer of IgG and IgA was observed by all of them, sharply in week 6 (P < 0.05). In all study groups, their ratio of IgG2a/IgG1 was upper 1, indicating Th1-biased responses. Wild-type viral neutralization assay showed that the secreted antibodies in vaccinated mice and recovered COVID-19 patients could neutralize SARS-COV-2 variants alpha and delta. After oral administration of oral vaccines, biodistribution assay was done. It was found that all of them had the same biodistribution pattern. The highest concentration of S-protein was seen in the small intestine, followed by the large intestine and liver.

The antibody titer. After immunization of BALB/c mice with saRNA LNPs, saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum alone, a high titer of IgG (Fig. 2a) and IgA ( Fig. 2b) was observed, sharply in week 6 (P < 0.05). Interestingly, the antibody titer of recovered COVID-19 patients was comparable with antibody titer of vaccinated mice at week 6 (P > 0.05). Another finding was that the production of antibody in vaccinated mice was dose and time dependent. To find Th1-biased responses in vaccinated mice, the ratio of IgG2a/IgG1 and was measured. In all study groups, their ratio of IgG2a/IgG1 was upper 1, indicating a Th1-biased response (Fig. 2c).
Viral neutralization assay. The secreted antibodies in vaccinated mice and recovered COVID-19 patients could neutralize SARS-COV-2 variants alpha (B.1.1.7) (Fig. 3a) and delta (B.1.617) (Fig. 3b). There were significant differences between the neutralization titer of all study groups and negative control (P < 0.05). Also, there were significant differences between neutralization titer in mice vaccinated with different dose of saRNA LNPs and transfected Lactobacillus plantarum LNPs (P < 0.05). Importantly, there was no significant difference  Figure 2. The IgG (a) and IgA (b) titer in mice vaccinated with saRNA LNPs, transfected Lactobacillus plantarum LNPs, and transfected Lactobacillus plantarum alone and in recovered COVID-19 patients against SARS-CoV-2. To find Th1-biased responses in vaccinated mice, the ratio of IgG2a/IgG1 was calculated (c). * indicates significance difference at P < 0.05 by one-way ANOVA with n = 10 biologically independent mice and n = 10 recovered COVID-19 patients. All data were shown as mean ± SD. www.nature.com/scientificreports/ between different formulation at the same dose (P > 0.05). The correlation analysis between SARS-CoV-2 specific IgG or IgA titer and SARS-CoV-2 neutralization titer can be seen in Supplementary 1.

IFN-γ ELISpots and the secretion of IL-6.
Splenocytes from mice immunized with saRNA LNPs, saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum alone had a high IFN-γ secretion with dose-dependent manner (Fig. 4a). Significant differences were seen between spleno-  The secretion of IL-6 (b) by re-stimulated splenocytes. * indicates significance difference with P < 0.05 using one-way ANOVA with n = 10 biologically independent mice. All data were shown as mean ± SD. www.nature.com/scientificreports/ cytes count in all immunized mice and negative control (P < 0.05). The secretion of IL-6 ( Fig. 4b) by re-stimulated splenocytes confirmed this data. Significant differences were also seen between the concentration of IL-6 produced by activated splenocytes in all immunized mice and negative control (P < 0.05). We also analyzed the serum level of IFN-γ, TNF-α, IL-4, and IL-10 in all vaccinated mice (supplementary 2).
Biodistribution assay. Biodistribution assay showed that saRNA LNPs (Fig. 5a), saRNA transfected Lactobacillus plantarum LNPs (Fig. 5b), and saRNA transfected Lactobacillus plantarum alone (Fig. 5c) had the same biodistribution pattern. The highest concentration of S-protein was seen in the small intestine, followed by the large intestine and liver. Significant differences was seen between the concentration of S-proein in small intestine and other organs (P < 0.05).

Discussion
In  19 . In our study, BALB/c mice were also orally vaccinated by saRNA transfected Lactobacillus plantarum LNPs and saRNA transfected Lactobacillus plantarum alone. Miraculously, we saw that both of them could also stimulate the immune system of mice, produce IgG and IgA, and neutralize SARS-COV-2.
In this study, we encapsulated saRNA molecules and transfected Lactobacillus plantarum by LNPs to escape the turbulent GIT environment. If we wanted to make a comparison and say which formulation had higher efficacy, we can say that saRNA LNPs was better than other formulation at the same dose. The saRNA transfected Lactobacillus plantarum LNPs and saRNA transfected Lactobacillus plantarum alone were in the next level. Based on biodistribution assay, saRNA LNPs, saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum alone had the same biodistribution pattern and the highest concentration of S-protein was seen in mice vaccinated with saRNA LNPs. It seems that all the formulations used in this study were able to pass through the stomach and reach to small and large intestines. It is a very valuable finding because these formulations may be used as an edible vaccine. The term of edible vaccines was first used by Charles Arendzen in 1990 to refer to any type of food that can stimulate the immune system and act as a vaccine against a specific disease 20 . In general, when a food product is ingested by GIT, both mucosal and humoral immune systems are stimulated 21 . Edible vaccines are genetically modified products that have an immunogenic component to produce antibody 22 . They have many advantages compared with traditional vaccines, such as lower manufacturing cost and side effects 23 . The production of traditional vaccines is very expensive and limited in most countries, but in contrast, the production, purification, sterilization, and distribution of edible vaccines are easier 24 . However, they have also some limitations because edible vaccines are still new and in development and more researches must be done before widespread human usage 21   www.nature.com/scientificreports/ Like all experimental studies, this study had some limitations. Here, there was no estimation on the durability of responses for antibodies and T-cell response. Also, there is no head to head comparison with "traditional" mRNA vaccine. These limitations must be considered in future studies. On the other hands, this study had significant scientific novelties, including: (1) the use of Norovirus sequence in the saRNA construct. Norovirus has two main structural proteins which can bind to different cells of gut 27 . Also, human norovirus also targets enteroendocrine epithelial cells in the small intestine 28 . It is the first report for COVID-19 vaccine and this formulation can be used for both oral and edible vaccine. (2) The use of saRNA transfected Lactobacillus plantarum LNPs and saRNA transfected Lactobacillus plantarum alone as novel oral vaccines against SARS-COV-2. It is the first report and this finding can help us to design edible vaccines against SARS-COV-2. In conclusion, the oral vaccines, based on saRNA LNPs, saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum alone, could induce a Th1-biased response to produce a high titer of IgG and IgA against SARS-CoV-2. The produced antibodies could neutralize SARS-CoV-2 variants alpha and delta. An important finding was that all formulations had the same biodistribution pattern and the highest concentration of S-protein was seen in small intestine of mice vaccinated with saRNA LNPs.

Encapsulation of linear saRNA and transfected Lactobacillus plantarum.
To encapsulate saRNA and transfected Lactobacillus plantarum, we used a simple chemical process 18 in which 100 1 µg purified saRNA and 10 6 CFU/mL of transfected Lactobacillus plantarum were separately mixed with an ethanolic lipid mixture of 1,2-dilinoleyloxy-3-dimethylaminopropane, 1,2-diastearoylsn-glycero-3-phosphocholine, cholesterol, and PEG-DMG 2000 at a ratio of 10:48:2:40. The mixture was stirred vigorously by a T-mixer and then placed in a dialysis bag to purify overnight. The synthesized LNPs were stored at 4 °C. The particle size distribution, zeta potential, polydispersity index (PDI) of LNPs were determined by a dynamic light scattering (DLS) (Malvern Instruments Ltd, Malvern, UK). For negative control plasmid, the same encapsulation process was also carried out.
In this study, vaccine administration was done by a needleless insulin syringe and the vaccine volume was 100 µL for each dose. Serum samples were collected at weeks 2, 4, and 6 and the spleens were removed at week 6. Here, the serum samples of recovered COVID-19 patients (n = 10, age = 40 ± 5, female/male = 60/40), suffered from SARS-COV-2 variant B.1.617 were also collected from Zahedan University of Medical Sciences, Zahedan, Iran following written informed consent (IR.ZAUMS.REC.1399.317 and IR.ZAUMS.REC.1399.316). All of them had grade II COVID-19 and they had been hospitalized for 2 weeks. One week after discharge from the hospital, Real-time PCR was done and their serum samples were collected. All recovered COVID-19 patients had negative PCR result at the time of sampling. Here, all experiments were under an approval of ethical committee of Zahedan University of Medical Sciences, Zahedan, Iran (IR.ZAUMS.REC.1400.071).
The evaluation of antibody titer. To evaluate antibody titer, an ELISA assay was used, according to Tian et al. 29 . In the first step, a high binding ELISA plates (Biomat, Italy) were coated with SARS-CoV-2 Spike Protein Recombinant Antigen (Sigma-Aldrich) at 1 mg/mL in PBS overnight at 4 °C. The plates were washed 3 times with PBS and blocked with 10% BSA (Sigma-Aldrich) and 3% sucrose (Sigma-Aldrich) at 4 °C overnight. Then, to inactivate blood complements, all serum samples were incubated at 56 °C for 30 min. The serum samples were separately diluted and 100 µL of each serum samples was added to the ELISA plate. The plates were incubated at 37 °C for 2 h and then were washed 3 times with PBS. After incubation and washing, they were separately incubated with secondary antibodies, including (1) anti-mouse and anti-human IgG-HRP (Southern Biotech), (2) anti-mouse IgG1-HRP (Southern Biotech), (3) anti-mouse IgG2a-HRP (Southern Biotech), (4) anti-mouse and anti-human IgA-HRP (Southern Biotech), with 1:5000 at 37 °C for one hour. After washing with PBS, 100 µL 3,3′, 5,5′-tetramethylbenzidine (TMB) substrate (Sigma-Aldrich) was added and incubated at 37 °C for 15 min. Then, 100 µL of sulfuric acid 1% (Sigma) was added and the optical density (OD) of each well was measured at 450 nm by a spectrophotometer (BioTek Industries). To set up ELISA cut-off values, 5 healthy mice without any vaccination and 5 healthy human which did not receive COVID-19 vaccine or suffered from COVID-19 were considered as negative control. The serum sample was considered positive when the OD was above the cut-off value. The cut-off value for mouse and human IgG was 0.140, for mouse and human IgA was 0.150, for mouse IgG2a and IgG1 was 0.155. Finally, the highest dilution titer which was above cut-off value was recorded.

Wild-type viral neutralization assay.
To evaluate the ability of sera to neutralize SARS-CoV-2 virus, wild-type viral neutralization assay was applied. Based on the article of McKey et al. 18 , SARS-CoV-2 variant B.1.1.7 and variant B.1.617 were isolated and cultured on Caco2 cells in DMEM (Gibco) containing 10% FBS (Gibco), 1% L-glutamine (Thermo Fisher Scientific), and 1% penicillin-streptomycin (Thermo Fisher Scientific) for 5 days at 37 °C. These variants were from clinical samples from reference laboratory of Zahedan University of Medical Sciences, Zahedan, Iran. Finally, they were purified by Caesium chloride. The serum samples were taken from vaccinated mice at week 6 and all of them were first incubated at 56 °C for 30 min and were serially diluted in DMEM (Gibco, Thermo Fisher Scientific) with 1% penicillin-streptomycin (Thermo Fisher Scientific), 0.3% BSA fraction V (Thermo Fisher Scientific) and 0.25 µg/mL trypsin (Worthington). Serum dilutions were separately incubated with 100 TCID 50 per well of SARS-CoV-2 variant B.1.1.7 and variant B.1.617 for 1 h at room temperature. Then, they were transferred to 96-well plates pre-seeded with HEK293T/17 cells and incubated at 37 °C for 5 days. After incubation, 100 µL of crystal violet (Sigma-Aldrich) was added to each well and scored the cytopathic effect. The neutralization titer was calculated as the reciprocal of the highest serum dilution at which full virus neutralization occurred.
To detect the level of IL-6, a high binding ELISA plate (Biomat, Italy) was separately coated with anti-mouse IL-6 (Southern Biotech) and then diluted samples from supernatant of activated mouse splenocytes were separately added. After 1 h incubation at 37 °C, plates were washed with PBS and then 100 μL of secondary antibody, including anti-mouse IL-6-HRP (Southern Biotech), was added. Then, 50 μL of TMB (Sigma-Aldrich) was added. After 15 min, 100 μL sulfuric acid (Sigma) was added and OD of each well was read by a Spectrophotometer at 450 nm (BioTek Industries) and then the serum level of IL-6 was quantified by standard curve 18 . Biodistribution assay. Biodistribution assay 31 was used to find out how saRNA LNPs, transfected Lactobacillus plantarum LNPs, and transfected Lactobacillus plantarum alone are distributed and expressed in different organs and tissue of mice. For this purpose, BALB/c mice aged 6-8 weeks were orally administered with 10 µg saRNA LNPs, 10 µg transfected Lactobacillus plantarum LNPs, and 10 µg transfected Lactobacillus plantarum. After 24 h, mice were sacrificed and sampled from their major organs and tissues, such as small intestine,