Neutralization of SARS-CoV-2 Variants of Concern Serum obtained at day 39 from hamsters vaccinated twice with MVA-S was pooled and tested for neutralization against SARS-CoV-2 VoC using a cytopathic effect (CPE)-based neutralization test (CPENT), as previously described (23). In brief, serial 2-fold dilutions (1:4, 1:8, 1:16, 1:32, 1:64, and 1:128) of pooled serum were incubated for 1 h at 37C with equal volumes of SARS-CoV-2 virus containing 100 TCID50 (prototypic strain B.1 and VoC B.1.1.7, B.1.351, P.1, B.1.167.2, and B.1.1.529). This mixture was then put onto a Vero E6 cell layer (2 104 cells/well in 96-well plates) and incubated for 3 days at 37C, after which the percentage of CPE was scored visually. Cell survival in each well relative to the mean of the virus control (VC) was calculated as follows: % live cells = (%CPEwell-%CPEVCmean)/(%CPECellControlmean-%CPEVCmean) 100. The median inhibitory concentration (IC50) of the serum was then obtained by non-linear curve fitting on the percentage of live cells as a function of the serum concentration. All assays were performed in triplicate.
RNA Extraction and Quantification of SARS-CoV-2 Subgenomic RNA by RT-qPCR RNA was extracted from 30 mg of homogenized lung tissue using the E.Z.N.A. Total RNA Kit I (Omega Bio-Tek), following the manufacturers instructions. SARS-CoV-2 subgenomic RNA copies (N gene) were quantified by RT-qPCR, as previously described (13). The relative fold change of SARS-CoV-2 subgenomic RNA levels was calculated by the 2-Cq method with -actin RNA levels for normalization.
Infectious Virus Titration For quantification of infectious SARS-CoV-2 viral particles, the supernatant of homogenized and centrifuged lung tissue was incubated on confluent Vero E6 cells. Infectious viral titers were calculated after 3 days by the method of Spearman and Krber (24) and expressed as TCID50 per 100 mg of homogenized lung tissue.
Lung Histopathology Lungs were fixed in 4% paraformaldehyde in PBS for a minimum of 24 h and then embedded in paraffin. Tissue sections of 4 m were stained with hematoxylin and eosin and scored for signs of lung damage by an expert pathologist, as previously described (18). Scores of 03 were given for the following parameters: intra-alveolar edema, lymphoid follicles, apoptotic bodies in the bronchi walls, necrotizing bronchiolitis, perivascular edema, bronchopneumonia, perivascular cuffing, peribronchiolar inflammation, vasculitis, and perivascular inflammation.
Statistical Analysis All statistical evaluations were performed with GraphPad Prism Version 9.1.2 (GraphPad Software, Inc.). Data are presented as means SEM. Statistical significance between conditions was calculated using the non-parametric MannWhitney test (significance at p-values <0.05).
Results MVA-S Vaccination in Hamsters Elicited S-Specific Antibodies That Neutralized SARS-CoV-2 Variants of Concern
To assess the ability of the MVA-S vaccine candidate to elicit SARS-CoV-2-specific binding and nAbs, groups of female Syrian hamsters (n = 12 per group) were immunized in a one- or two-dose regimen with MVA-S (Figure 1A). One group of animals received a prime dose of 2 107 PFU of MVA-S via the i.p. route on day 0, followed by a booster dose with 4 107 PFU of MVA-S at day 21 (MVA-S/MVA-S), whereas a second group only received a single dose of 4 107 PFU of MVA-S at day 21 (/MVA-S). A third group primed and boosted with similar doses of MVA-WT at days 0 and 21 served as the control group (MVA-WT/MVA-WT) (Figure 1A). Vaccination with MVA-S or MVA-WT had no effect on body weight progression ( Figure S1A ).
For serological analysis, serum was collected at days 21 (before boosting; 21 days post prime) and 39 (before SARS-CoV-2 challenge; 18 days post boost). First, we analyzed the presence of anti-S and anti-RBD-binding IgG antibodies at day 39 by ELISA. The results showed that single and double MVA-S vaccination elicited similarly high IgG titers against the S protein (Figure 1B) and more specifically against the RBD (Figure 1C). These results were also confirmed by an IIFA assay against the S protein, with induction of high titers of binding antibodies at 21 days post prime MVA-S immunization ( Figure S2A ) that were further enhanced after the booster dose ( Figure S2B ).
Next, the evaluation of the levels of SARS-CoV-2 nAbs at day 39 by using a live microneutralization assay showed that a single and double dose of MVA-S elicited high titers of nAbs against SARS-CoV-2 (MAD6 strain, containing D614G mutation in the S protein) (Figure 1D), with significantly higher titers in the two-dose MVA-S regimen compared to one dose of MVA-S and with no neutralizing activity observed in the MVA-WT control group. Additionally, a neutralization assay using S-pseudotyped VSV particles yielded similar results, with induction of high nAb titers already at 21 days after the first MVA-S dose ( Figure S2C ) that were further enhanced after the booster dose ( Figure S2D ), although in 2 out of 12 animals, no nAbs were detected with the S-VSV pseudovirus ( Figure S2D ).
To address the increasing clinical importance of SARS-CoV-2 VoC and neutralizing capacity of the MVA-S vaccine, pooled serum samples obtained at day 39 from double-vaccinated hamsters were tested for neutralization against SARS-CoV-2 VoC alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.167.2), and omicron (B.1.1.529) and compared to the prototypic SARS-CoV-2 B.1 strain (Figure 1E). As expected, the median inhibitory concentration (IC50 SEM) of serum was the lowest for neutralization of the prototypic B.1 strain (5.14 0.52 10-3), shortly followed by that of the alpha (7.30 1.06 10-3), gamma (8.72 1.95 10-3), and delta (1.14 0.11 10-2) strains. The beta and omicron VoCs could also be neutralized, although at higher serum concentrations (2.83 0.54 10-2 for beta and 5.48 0.23 10-2 for omicron).
MVA-S Vaccination Prevented SARS-CoV-2 Replication and Lung Pathology in Hamsters Three weeks after the last vaccine dose (day 42), all hamsters were infected intranasally with 2 105 TCID50 of SARS-CoV-2 (Figure 1A). Initially, body weight was analyzed after challenge, and hamsters from all groups experienced a similar slight drop in body weight after SARS-CoV-2 infection, but by day 4 post-challenge, vaccinated groups had significantly recovered compared to control animals ( Figure S1B ). At 2, 4, and 14 dpi, four animals per group were sacrificed, and their lungs were analyzed for signs of SARS-CoV-2 replication and virus-induced lung damage. MVA-S vaccination resulted in reduced levels of SARS-CoV-2 subgenomic (sgm) RNA (N gene) in double-vaccinated hamsters already at 2 dpi (Figure 2A). At 4 dpi, all MVA-S-vaccinated animals, even after a single dose, had approximately a significant 103-fold reduction of SARS-CoV-2 sgmRNA levels in their lungs compared to the MVA-WT control group (Figure 2A). At 14 dpi, overall viral sgmRNA levels had gone down but were still significantly reduced in hamsters vaccinated with one or two doses of MVA-S (Figure 2A). Infectious virus titers were consistently reduced in lungs of vaccinated animals at 2 and 4 dpi (Figures 2B, C), and at 4 dpi, even up to a significant 105-fold reduction, with 2 out of 4 animals in each vaccinated group having no detectable virus (Figure 2C). At 14 dpi, the infectious virus had disappeared from the lungs in all groups (Figure 2D).