Extended Data Fig. 4. Immunization with OTS4-5, OTS7-8, OTS4-5-7-8, and OTS-206 protects K18-hACE2 mice and Syrian hamsters from SARS-CoV-2 infection.
a, K18-hACE2 transgenic mice (7-16 weeks of age, n=8 mice/group) were immunized with either OTS viruses and SARS-CoV-2 WT or mock medium. Blood samples were taken on day 15 post-immunization. At 21 days post-immunization, mice were challenged with SARS-CoV-2 WT and euthanized on days 5 and 14 post-challenge to obtain organ samples (nose, lungs, brain, and olfactory bulbs) for evaluation. Overview created with BioRender.com. b, Clinical symptoms over the course of infection (mean +/- SEM) and c, genome copy numbers (genome equivalence per ml, gEq/mL) oropharyngeal swabs and in post challenge samples ((d, orophayngial swabs, e, brain and olfactory bulb (OB) 5-6 dpc) and g, brain, OB, nose and lung 14 dpc).f, h, Infectious virus titers from the brain, lung and nose samples. i, Histopathological lung scores at 14 dpc. j, Hematoxylin and eosin stain of lung sections (n=4 per group and time point). Pneumonia (asterisk), perivascular and peribronchiolar cuffings (arrowhead), tertiary lymphoid follicle formations (arrow). Scale bar, 500 μm. k, Virus neutralization test of 15 dpi (pre-challenge) and 5 and 14 dpc (post-challenge) sera against WT. l, Share of CD8+ T cells reacting to spike-stimulated condition. Workflow is given in Supplementary Fig. 1 and in the methods section. m, Sera samples from OTS4-5 or OTS7-8 vaccinated Syrian hamsters challenged with SARS-CoV-2 WT (Fig. 3a), as well as sera of co-housed contact animals, were analyzed and tested positive by SARS-CoV-2-RBD specific ELISA. n, Additionally, virus neutralizing capacity was confirmed for these samples, while mock, OTS4-5 contact, and OTS7-8 contact animals showed only low titers. o, Organ samples of 14 dpc of OTS-206 vaccinated and subsequently SARS-CoV-2 Omicron BA.2 challenged Syrian hamsters (Fig. 3f) were analyzed by RT-qPCR. p, Serological evaluation by SARS-CoV-2-RBD specific ELISA confirmed transmission of BA.2 challenge virus to the naïve contact animals for the mock vaccinated as well as for the OTS-206 vaccinated animals. q, Live virus neutralizing capacity comparison showed substantial neutralizing titers of the OTS-206 vaccinated animals against ancestral SARS-CoV-2 and Omicron BA.2 VOC. r, Pneumonia-induced pulmonary atelectasis at 5 dpi was assessed, and s, histopathology images showed atelectasis. Scale bar, 2.5 mm. t, Virus antigen score and u, immunohistochemistry for SARS-CoV nucleocapsid protein detection were conducted. Scale bar, 100 µm. Statistical significance was assessed by two-sided, unpaired, nonparametric multiple t-test with Mann-Whitney test (compared ranks) (panel d-f, i, k, n and q). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. No asterisk indicates no statistical significance.