Journal: bioRxiv

Article Title: Ending transmission of SARS-CoV-2: sterilizing immunity using an intranasal subunit vaccine

doi: 10.1101/2022.07.14.500068

Figure Lengend Snippet: (A) 3D structure of trimeric S protein (B.1.351) with the twelve mutations indicated (PDB: 7VX1) . (B) Study timeline: We immunized Balb/c mice (n=5/group) with a single dose of NanoSTING-S intranasally (IN) followed by the collection of serum every week. We monitored the bodyweights of the animals every week after the immunization. We euthanized the animals at day 28 and then collected BALF, serum, lungs, and spleen. Bodyweight change, ELISA (IgG & IgA), and ELISPOT (IFNγ and IL4) were used as primary endpoints. Naïve Balb/c mice were used controls (n=4/group). (C-F) Humoral immune responses in the serum and BALF were evaluated using S-protein-based IgG & IgA ELISA. (G, H) Splenocytes (G) or lung cells (H) were stimulated ex vivo with overlapping peptide pools, and IFNγ & IL4 responses were detected using an ELISPOT assay. (I) Experimental set up for challenge study in hamsters: We immunized Syrian golden hamsters (n=10/group) intranasally with two doses of NanoSTING-S (first dose at day -42 and second dose at day -18, and challenged the hamsters intranasally with 3 × 10 4 CCID 50 of the SARS-CoV-2 Delta VOC on day 0. Post challenge, we monitored the animals for 6 days for changes in the bodyweight. We euthanized half of the hamsters on day 2 and other half at day 6 for histopathology of the lungs, with viral titers of lung and nasal tissues measured on day 2 and day 6. (J) Percent bodyweight change of hamster compared to the baseline at the indicated time intervals. (K, L) Viral titers measured by plaque assay in lungs and nasal tissues post day 2 and day 6 of infection. The dotted line indicates the limit of detection of the assay (LOD). (M, N) Pathology score and a representative hematoxylin and eosin (H&E) image of the lung showing histopathological changes in hamsters treated with NanoSTING-S and PBS; all images were acquired at 10x & 20×; scale bar, 100 µm. For ELISA, ELISPOT, viral titers and lung histopathology data, analysis was performed using a Mann-Whitney test. Vertical bars show mean values with error bar representing SEM. Each dot represents an individual hamster or mouse. Weight data was compared via mixed-effects model for repeated measures analysis. Lines depict group mean bodyweight change from day 0; error bars represent SEM. Asterisks indicate significance compared to the placebo-treated animals at each time point . ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns: not significant. See also , &

Article Snippet: Briefly, (i) NanoSTING-S vaccine was prepared by gently mixing 10µg of trimeric spike protein-B.1.351 (Acrobiosystems, #SPN-C52Hk) with 20µg of NanoSTING. (ii) NanoSTING-N (Wuhan) (BEI, # NR-53797): Two different concentrations of the Nucleocapsid protein were taken: NanoSTING-N10 (10 µg of N protein) and NanoSTING-N20 (20 µg of N protein) were mixed separately with 20 µg of the NanoSTING. (iii) NanoSTING-N: 20µg of nucleocapsid protein-B.1.17 (Acrobiosystems, #NUN-C52H8) was mixed with 20µg of NanoSTING. (iv) NanoSTING-NS: 10µg of trimeric spike protein-B.1.351 (Acrobiosystems, #SPN-C52Hk) and 20µg of nucleocapsid protein-B.1.17 (Acrobiosystems, #NUN-C52H8) were mixed with 20µg of NanoSTING.

Techniques: Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Ex Vivo, Histopathology, Plaque Assay, Infection, MANN-WHITNEY