key: cord-0926281-zciugjtx authors: Mohammadi, Golamabbas; Sotoudehnia Koranni, Zahra; Jebali, Ali title: The oral vaccine based on self-replicating RNA lipid nanoparticles can simultaneously neutralize both SARS-CoV-2 variants alpha and delta date: 2021-10-07 journal: Int Immunopharmacol DOI: 10.1016/j.intimp.2021.108231 sha: 2c853eda7fd9185893504cc342b7565dee92763a doc_id: 926281 cord_uid: zciugjtx The aim of this study was to evaluate self-replicating RNA lipid nanoparticles (saRNA LNPs) to neutralize SARS-CoV-2 variants delta (B.1.617 lineage) and alpha (B.1.1.7 lineage). Before immunization of mice with saRNA LNPs, we saw high expression of S-protein at both mRNA and protein levels after transfection of HEK293T/17 cells with saRNA LNPs. After oral immunization of BALB/c mice with 0.1 – 10 µg saRNA LNPs , a high quantity of SARS-CoV-2 specific IgG and IgA antibodies were seen with a dose-dependent pattern. Importantly, the ratio of IgG2a/IgG1 in serum of vaccinated mice showed Th1/Th2 skewing response. We also found that the secreted antibodies could neutralize SARS-CoV-2 variants delta (B.1.617 lineage) and alpha (B.1.1.7 lineage). Re-stimulated splenocytes of vaccinated mice showed high secretion of IFN-γ, IL-6, and TNF- α . The authors think that although the preclinical study confirmed the efficacy of saRNA LNPs against SARS-CoV-2, the actual efficacy and safety of the oral vaccine must be evaluated in clinical trials. Oral vaccine; Self-replicating RNA; B.1.1.7 lineage; B.1.617 lineage Vaccines have different formulations and they are becoming more advanced every day [1] . The use of nucleic acids and nanoparticles are two examples of advanced materials, recently used in vaccine production [2] . Early studies focused on the use of DNA instead of RNA because RNA is less stable than DNA [3] . DNA vaccines produced poor results in human clinical trials [4] and this led to using RNA. Of course, this change in strategy was attributed to the success of cancer immunotherapy by mRNA molecules [5, 6] . Moreover, the success of developing mRNA-based COVID-19 vaccines opens a new window to vaccine design [7, 8] . There are currently two types of RNA vaccines, including conventional mRNA and self-replicating RNA (saRNA) [9] . These RNAs are produced in vitro and encode pathogen antigens [10] . To produce a saRNA, the sequence of an RNA-dependent RNA polymerase (RDRP) complex from viral origin to amplify saRNA, as well as the 5´ and 3´ untranslated regions (UTR), is needed for its replication in cytoplasm [11] . After expression of saRNA, activation of immune system is occurred [12] . The production of saRNA vaccines is not very complicated and can be easily integrated into a production line [13] . For example, Hekele et al. designed and produced saRNA vaccine for influenza H7N9 within eight days [14] . There are several formulations that can be used to deliver saRNA vaccines, including cationic polymers [15] , lipopolyplexes [16] , and lipid nanoparticles (LNPs) [17] . Interestingly, because of the self-replicating property of saRNA, a high immune response can be achieved with low doses of saRNA vaccines [18] . Importantly, the site of action for saRNA is the cytoplasm and it does not require to enter the cell nucleus. Indeed, there is no risk of integration of saRNA into the genome [5, 6, 19] . Although a variety of COVID-19 vaccines have been developled and some are being used globally, the majority of the population of developing countries still have not been vaccinated because of lack of funds, no infrastructure, and social issues [20] . They need a simple and inexpensive vaccine. Therefore, we sought to design, produce, and evaluate an oral vaccine that could be easily used. The aim of this study was to evaluate saRNA LNPs in mice to neutralize SARS-CoV-2 variants delta (B.1.617) and alpha (B.1.1.7) variants. To synthesize the saRNA construct, a common bacterial plasmid vector with a T7 promoter was used. The required sequence for saRNA construct was synthesized and sub-cloned by Biomatik, The plasmid encoding saRNA construct was transformed into E. coli (institute Pasteur, Iran), cultured in Luria Broth with 100 μg/mL carbenicillin (Sigma Aldrich, UK). Plasmids were purified using a Plasmid Plus MaxiPrep kit (QIAGEN, UK) and their concentration and purity were measured on a NanoDrop spectrophotometer (ThermoFisher, UK). Then, cloned plasmids were linearized using MluI for 3 h at 37 °C. Then, saRNA transcripts were produced using 1 μg of linearized DNA template in a MEGAScript™ reaction (Ambion, UK) for 1 h at 37 °C. One μg linear saRNA was mixed with 1 μM ScriptCap™ (CellScript, WI, USA) for 1 h at 37 °C. Synthesized saRNA was purified by LiCl precipitation, re-suspended in RNA storage buffer, and stored at −80 °C. To evaluate the purity of synthesized saRNA, the A260/A280 ratio was measured using the NanoDrop spectrophotometer (ThermoFisher, UK) [21] . To encapsulate saRNA, we used a simple chemical process [21] in which 0.1, 1, and 10 μg of IR.SIRUMS.REC.1400.001). We used 10 BALB/c mice aged 6-8 weeks in each study group and they were orally immunized with 0.1, 1, and 10 µg saRNA LNPs at weeks 1 and 3. For each mouse, 100 µL of vaccine mixture was orally administered using a needle-free insulin syringe. In the control groups, BALB/c mice were orally immunized with 10 µg positive control LNPs and 10 µg negative control LNPs at weeks 1 and 3 as described. Serum samples were collected at weeks 2, 4, and 6 and the spleen of vaccinated mice was removed at week 6. Here, the serum samples of recovered COVID-19 patients (n=10) were obtained from Zahedan University of Medical Sciences, Zahedan, Iran. All of them had been infected with the delta variant. Written informed consent was given from all participants (ethical codes: IR.ZAUMS.REC.1399.317 and IR.ZAUMS.REC.1399.316). All recovered patients showed a negative PCR test at the time of sampling. A semi-quantitative ELISA was used to determine the levels of IgG, IgG1, IgG2a, and IgA antibodies in the sera of vaccinated mice. Also, the levels of IgG and IgA antibodies were determined in the sera of recovered COVID-19 patients. First, high binding ELISA plates (Biomat, Italy) were coated with SARS-CoV-2 S-protein recombinant antigen (Sigma-Aldrich) at 1 μgmL −1 . After washing plates, 50 μL of diluted serum samples collected from immunized mice and recovered COVID-19 patients were separately added to wells. After incubation for 1 h at 37 °C, plates were washed with PBS and then 100 μL of the following secondary antibody was separately added: 1) anti-mouse IgG-HRP, 2) anti-mouse IgG1-HRP, 3) anti-mouse IgG2a-HRP, 4) anti-mouse IgA-HRP, 5) anti-human IgG-HRP, 6) antihuman IgA-HRP (Southern Biotech). After incubation and washing with PBS, 50 μL of 3,3′, 5,5′tetramethylbenzidine was added and then the reactions was stopped by adding 50 μL of 10% sulfuric acid. Finally, the absorbance of each well was read by a Spectrophotometer at 450 nm (BioTek Industries) and serum levels of antibodies was measured using a standard curve. To evaluate the ability of vaccinated mice or recovered COVID-19 patients to neutralize SARS-CoV-2 virus, wild-type viral neutralization assay was applied according to McKey et al [21] . GraphPad Prism (version 8.4) was used to prepare graphs and statistics. One-way ANOVA was used and P values less than 0.05 were considered significant (with n =10 biologically independent mice and n=10 recovered COVID-19 patients). All data are shown as mean± standard deviation (SD). Synthesized linear saRNA was encapsulated in LNPs and then characterized. The average particle size and zeta potential of saRNA LNPs were 100±5 nm and +22±0.6 mV, respectively (Figure 1d and Figure 1e) . The highest entrapment efficiency of saRNA was 67±2 %. Before immunization of mice with saRNA LNPs, their efficacy was verified in HEK273T/17 cells. High expression of S-protein was observed at both mRNA and protein levels after transfection of HEK293T/17 cells with saRNA LNPs (Figure 1f and Figure 1g) . It was found that the relative expression of S-protein was increased with increasing LNP dose. BALB/c mice were orally immunized with 0.1-10 µg saRNA LNPs at weeks 1 and 3 and their serum samples were then collected at weeks 2, 4, and 6. ELISA assay showed a high quantity of SARS-CoV-2 specific IgG, IgG1, IgG2a, and IgA antibodies in a dose-dependent manner in serum of mice (Figure 2a-c) . Interestingly the levels of IgG and IgA antibodies in mice immunized with 10 μg saRNA LNPs was almost close to antibody levels in recovered COVID-19 patients (P>0.05). Here, significant differences were found between the level of antibodies in vaccinated mice or variants. Then, they were transferred to pre-seeded well with HEK293T/17 cells and incubated for 5 days. After incubation, the cytopathic effect was recorded and the neutralization titer was calculated. We observed a high viral neutralization titer in mice vaccinated with saRNA LNPs at 0.1-10 μg in a linear dose-dependent manner. We also found that the secreted antibodies induced by saRNA LNPs could neutralize both SARS-CoV-2 variants B.1.1.7 (alpha) and B.1.617 (delta) (Figure 3a-b) . Interestingly, secreted antibodies detected in recovered COVID-19 patients could neutralize both variants. A significant positive correlation was observed between SARS-CoV-2 specific IgG and SARS-CoV-2 neutralization titer in both vaccinated mice and recovered COVID-19 patients (Figure 3c-d) . Splenocytes of vaccinated mice were first activated with SARS-CoV-2 peptides and then added to anti-IFN-γ-pre-coated plates. After incubation for overnight at 37 °C, detection antibodies, streptavidin-enzyme conjugate, and substrate were added. Finally, each well was examined under an optical microscope and the number of stained cells was calculated. We found that re-stimulated splenocytes of vaccinated mice had a high IFN-γ secretion in a linear dose-dependent manner (Figure 4a ). There was a significant difference between mice vaccinated with 10 μg saRNA LNPs and other groups (P<0.05). We also measured the secretion of IL-6 and TNF-α by re-stimulated splenocytes and the serum levels of IL-6 and TNF-α in vaccinated mice and recovered COVID-19 patients. It was shown that in both supernatants of re-stimulated splenocytes and vaccinated mouse sera, there was a high level of IL-6 and TNF-α in a dose-dependent manner (Figure 4b-e) . Here, significant differences were observed between mice vaccinated with 10 μg saRNA LNPs and other vaccinated groups (P<0.05). We found significant differences between the level of IL-6 and TNF-α in the serum of recovered COVID-19 patients compared with vaccinated mice by saRNA LNPs (P<0.05). In this study, we sought to develop a saRNA-based oral vaccine that could simultaneously fight both the SARS-COV-2 variants B.1.1.7 (alpha) and B.1.617 (delta). Vaccines based on saRNA have already been used for some infectious diseases [14, 22] . This technology has also been studied for COVID-19, showing some advantages versus conventional mRNA [21, 23] . The strength of saRNA-based vaccines is that they can induce potent immune responses at low doses because of their self-replicating property. Of course, these vaccines, like other vaccines, may have side effects and must be evaluated. Currently, although various vaccines against SARS-COV-2 are designed with different platforms [24] , most people of un-developed and developing countries still have not been vaccinated, which can be due to various reasons, such as economic and social problems [20] . Unfortunately, the genomic structure of SARS-COV-2 is not stable and it is susceptible to mutations [25] . This phenomenon leads to a significant reduction of vaccine efficacy with any platform [26] . In this study, the designed saRNA had alphavirus replicase, RDRP, as well as the gene coding for the S-protein. The replicase was required for saRNA amplification and mediates the production of this RNA [27] . This leads to the production of a large number of S-proteins that be taken up by antigen presenting cells (APCs). Of course, it should be noted that saRNAs, in order to efficiently be delivered to cells, must be protected. One of the best ways is to use LNPs [21] and they can be optimized for target cells [28] . We first tested the efficacy of saRNAs in HEK293T cells and then in mice. The high expression of S-protein at both mRNA and protein levels was observed after transfection of HEK293T/17 cells. This valuable finding gave us hope that the saRNA LNPs might stimulate the immune system and could act as an effective vaccine. In this study, the vaccine was orally administered because our goal was to stimulate the immune cells located in the mucosa. Oral administration is much easier than injectable vaccines and is especially more acceptable by Neutralizing titers after heterologous prime-boost were at least comparable or higher than the titers measured after homologous prime-boost vaccination with viral vectors [23] . It is important to know the intestinal immune responses against SARS-CoV-2 is more effective than others [29] . Some studies on COVID-19 animal models [30] and COVID-19 patients with gastrointestinal symptoms [31] revealed that intraepithelial CD8 + lymphocytes and lamina propria residing CD4 + and CD8 + effector T cells are significantly expanded compared with healthy controls [32] . Importantly, inflammatory dendritic cells are significantly reduced in the lamina propria of COVID-19 patients with gastrointestinal symptoms [32] . These data suggest that intestinal infection with SARS-CoV2 alters immune signatures and leads to a more favorable immune response. Interestingly, the serum levels of IL-6 and IL-17 are lower in COVID-19 patients with GI symptoms compared with COVID-19 patients without GI symptoms. Another interesting point is that the presence of the virus in the GI tract may trigger a long-term production of anti-viral IgA antibodies, compared with IgG and IgM antibodies [33, 34] . Taken together, the oral vaccine, based on saRNA LNPs, could induce a Th1-biased response to produce a high quantity of SARS-CoV-2 specific IgG and IgA antibodies. We also found that the produced antibodies could neutralize SARS-CoV-2 variants B.1.1.7 (alpha) and B.1.617 (delta). All participants provided written informed consent for participation in the study before enrollment. The study was approved by review board of Sirjan School of Medical Sciences, Sirjan, Iran. The data that support the findings from this study are available from the corresponding author on reasonable request. The size distribution (d) and zeta potential (e) of saRNA LNPs by DLS apparatus. HEK293T/17 cells were treated with saRNA LNPs at 0.1-10 μg and the expression of S-protein was evaluated by Real-time PCR (f) and ELISA (g). All data were shown as mean±SD. * indicates significance difference with P<0.05 when compared with negative controls using a one-way ANOVA adjusted for multiple comparisons with n =5. The ratio of IgG2a/IgG1 was used to find Th1/Th2 skewing responses in vaccinated mice (d). * indicates significance difference at p < 0.05 when compared with negative control using a one-way ANOVA adjusted for multiple comparisons with n =10 biologically independent mice and recovered COVID-19 patients. . Splenocytes of vaccinated mice re-stimulated with SARS-CoV-2 peptides had a high IFN-γ secretion in a linear dose-dependent manner (a). The secretion of IL-6 (b) and TNF-α (c) by re-stimulated splenocytes. 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The full length sequence of saRNA construct used in this study The details of Real-time PCR and ELISA methods to evaluate the expression of S-protein The authors state no conflict of interest. Informed consent was obtained from all individuals included in this study.