key: cord-0911277-2cb8mn0d authors: Jawalagatti, Vijayakumar; Perumalraja, Kirthika; Hewawaduge, Chamith; Yang, Myeon Sik; Park, Ji-Young; Oh, Byung Kwan; Lee, John Hwa title: Bacteria-enabled oral delivery of a replicon-based mRNA vaccine candidate protects against ancestral and delta variant SARS-CoV-2 date: 2022-02-03 journal: Mol Ther DOI: 10.1016/j.ymthe.2022.01.042 sha: 4afd9f68ff7220cb974e1211f3742bbca4faf5fe doc_id: 911277 cord_uid: 2cb8mn0d The ongoing SARS-CoV-2 evolution has resulted in many variants, contributing to the striking drop in vaccine efficacy and necessitated the development of next-generation vaccines to tackle antigenic diversity. Herein we developed a multivalent Semliki Forest virus replicon-based mRNA vaccine targeting the receptor binding domain (RBD), heptad repeat domain (HR), membrane protein (M) and epitopes of nsp13 of SARS-CoV-2. The bacteria-mediated gene delivery offers the rapid production of large quantities of vaccine at a highly economical scale and notably allows the needle-free mass vaccination. A favourable Th1 dominated potent antibody and cellular immune responses were detected in the immunized mice. Further, immunization induced strong cross-protective neutralizing antibodies (NAbs) against the B.1.617.2 delta variant (Clade G). We recorded a difference in induction of IgA response by the immunization route with the oral route eliciting a strong mucosal sIgA response, which possibly has contributed to the enhanced protection conferred by the oral immunization. Hamsters immunized orally were completely protected against the viral replication in the lungs and the nasal cavity. Importantly, the vaccine protected the hamsters against SARS-CoV-2-induced pneumonia. The study provides proof-of-principle findings for the development of a feasible and efficacious oral mRNA vaccine against SARS-CoV-2 and its variants. As tools do not exist for hamsters, we evaluated the humoral and cellular immune responses 136 in BalB/c mouse immunized via the intramuscular and oral routes. The intramuscular 137 injection consisted of a single dose of 1×10 7 colony-forming units (CFU) . Whereas, the oral 138 route of administration consisted of two doses of 1×10 8 CFU at a 2-week interval. The 139 immune response was evaluated 3 weeks after the final immunization, and, at this point, all 140 mice receiving the vaccine had seroconverted with robust antigen-specific IgG, IgG1 and 141 IgG2a ( Figure 3A ). An IgG and IgG2a titre of >10 4 was recorded for RBD and HR, whereas 142 it was >10 3 for IgG1. Similarly, IgG, IgG1 and IgG2a titres of 10 3 to 10 4 were observed for 143 M and nsp13 proteins. Further, the ratio of IgG2a to IgG1 revealed a Th1 dominated immune 144 response ( Figure 3B ) 145 To assess the cellular immune response, splenocytes were stimulated with individual 146 recombinant proteins as a measure of recall immunity. We determined the changes in 147 CD3 + CD4 + and CD3 + CD8 + T cell subpopulation by FACS. Flow cytometric data were 148 analyzed by gating lymphocytes (P1) to identify CD3 + T cells (P2) and from P2, the 149 percentage of T cells expressing CD4 and CD8 was measured (Supplementary Figure 2) . 150 Immunization resulted in a significant increase in the proportions of CD4 + and CD8 + T cells 151 in response to protein stimulation than the placebo controls ( Figures 3C and 3D) . The 152 highest expansion in the CD4 + T cells was recorded for RBD whereas, M protein resulted in 153 the maximum increase in CD8 + T cells. Intracellular cytokine staining revealed a higher 154 number of IFN-γ positive CD4 + and CD8 + T cells in splenocytes from immunized mice 155 stimulated with respective proteins (Figures 3E and 3F) . Contrarily, very few cells (<0.1%) 156 expressing IL-4 were detected in the stimulated splenocytes (data not shown). Further, the intramuscular and oral routes ( Figure 3G ). The Th1 dominated cellular response was further 160 substantiated by an IFN-γ ELISpot assay. We detected a significantly higher number of secreting cells in response to stimulation of splenocytes with respective vaccine immunogens 162 ( Figure 3H) . The results support the elicitation of strong humoral and cellular immune 163 responses with polarization towards antiviral Th1 immunity by the replicon-based mRNA 164 vaccine. seroconverted with high titres of antigen-specific IgG (Figures 4B and 4C ). We recorded a 177 titre of >10 4 for RBD, HR and two animals in the oral group evidenced a titre of ≥10 5 . Whereas a titre of >10 3 was observed for M and nsp13 proteins. The multivalent vaccine was designed to elicit broad neutralizing antibodies (NAbs) 180 against SARS-CoV-2. To test the hypothesis, we next analyzed the ability of hamster sera to We detected the sIgA with a titre of >200 in the oral group whereas, no sIgA was detected in The raise of SARS-CoV-2 variants with mutations in the spike protein, particularly 282 the RBD and N-terminal domain (NTD) has been a great concern due to their ability to evade The authors declare that they have no competing interests. Copper-impregnated three-layer mask efficiently inactivates SARS-CoV2 Quantum dot-conjugated SARS-CoV-2 spike 695 pseudo-virions enable tracking of angiotensin converting enzyme 2 binding and 696 endocytosis Highly susceptible SARS-CoV-2 699 model in CAG promoter-driven hACE2-transgenic mice CAPITO-a web server-based 701 analysis and plotting tool for circular dichroism data SARS-CoV-2 parental strain and (E) SARS-CoV-2 B.1.617.2 variant. The dashed line in (D) E) represents the lower limit of detection (LLOD) Medium only control; VO-Virus only control; RBD-Receptor binding domain 779 repeat domain; M-Membrane glycoprotein; nsp13-Non-structural protein 13 Cytopathic effect; IFA-immunofluorescence assay B.1.617.2-delta variant SARS-CoV-2 NAb-Neutralizing antibody; CFU-Colony-forming units Data information: Data in (A) was analyzed by two-way ANOVA using Bonferroni post-test Data in (B and C) was analyzed by unpaired student's t-test. Data in (D and E) was analyzed 784 by one-way ANOVA using Bonferroni's Multiple Comparison Test. The data points represent 785 the individual value from each animal and error bars denote the SEM at 95% CI Figure 5. Evaluation of protection conferred by the vaccine in a hamster model 788 The intramuscular 789 injection consisted of a single dose of 2×10 7 CFU. Whereas, the oral route of administration 790 consisted of two doses of 2×10 8 CFU at a 2-week interval. Hamsters were challenged 3 791 weeks after the final immunization with 1×10 4 PFU of either parental strain (Clade L) or 792 delta variant (Clade G). Animals were sacrificed on day 5 post-challenge, and lung samples 793 were collected for virological and histopathological analysis A and D) Hamster body weights following viral challenge. (B-C) and (E-F) Lung viral 796 measurements in hamsters challenged with parental SARS-CoV-2 and B.1.617.2 SARS-CoV-797 2, respectively. (G) Viral load in the nasal wash of hamsters SARS-CoV-2. Data were presented as log10-transformed PFU/mL and viral N gene copies/g. The dashed line represents the lower limit of quantification (LLOQ)