Fig. 5.

Challenge of transgenic mice following prime and boost vaccinations. (A) Thirty K18- hACE2 mice (female, 7 wk old) were vaccinated IM with 2 × 10⁷ PFU of parental MVA or rMVA on days 0 (prime) and 21 (boost), and six remained unvaccinated. Six of the vaccinated mice were inoculated with parental MVA, two groups of six with 2P, and two groups of six with Tri. One 2P and one Tri group were boosted with the homologous vector and the other with RBD protein. Mice were bled on days 0, 21, and 35 to determine S binding and CoV-2 neutralizing titers. Naive mice and vaccinated mice 2 wk after boosting were infected IN with 10⁵ TCID50 of CoV-2. Mice were followed for weight loss and signs of morbidity. Two mice from each group were euthanized on days 2 (*) and 5 (**) after challenge to determine infectious CoV-2 virus and subgenomic mRNA. A second IN challenge of surviving mice and two added naive mice was performed 2 wk after the first challenge. (B) Binding antibody was determined by ELISA and plotted as 1/end point dilution. Dotted line indicates limit of detection. (C) Neutralizing antibody was determined by a pseudovirus assay and plotted as NT50. (D) Weights of surviving mice were determined daily and plotted as percent of starting weight. Symbol † indicates number of mice that died or were euthanized on a specific day. Data for one naive mouse was from a preliminary experiment. (E) Weights were determined after the rechallenge of surviving mice and two naive mice. (F) Virus titers in lung homogenates obtained on days 2 and 5 were determined by end point dilution and plotted as TCID50 per gram of tissue. The lower two data points for naive mice on day 2 were from a preliminary experiment. (G) Virus titers in nasal turbinate on days 2 and 5 were determined as in F and plotted as TCID50 per sample. (H) RNA was isolated from lung homogenates on days 2 and 5. CoV-2 N and S subgenomic RNAs were determined by ddPCR and plotted as copies per microgram of RNA. (I) RNA was isolated from nasal turbinate homogenates as described in H. Significance (P < 0.004) was determined by combining the values for N mRNAs or S mRNAs on day 2 from both control groups and comparing that to the combined values from rMVA vaccinated mice using Student’s t test. (J) Cytokine and chemokine RNAs in day 2 lung homogenates were determined by ddPCR. Values for control and naive mice were plotted together, as were those for rMVAs that received homologous boosts. X1 refers to sera collected 3 wk after prime; X2 refers to sera collected 2 wk after homologous boost; RBD Pro in red refers to heterologous boost with RBD protein.