Fig 1.
Replication of B/HPIV3, B/HPIV3/S-6P, B/HPIV3/S-Delta-6P and B/HPIV3/S-Omicron-6P in hamsters.
(A) Genome map of the B/HPIV3 empty vector, B/HPIV3/S-6P expressing the S-6P stabilized version of the spike protein (S) of the ancestral SARS-CoV-2 strain (blue, [25,26], and of the newly-generated versions B/HPIV3/S-Delta-6P (green) and B/HPIV3/S-Omicron-6P (orange). BPIV3 N, P, M and L genes are in white and HPIV3 F and HN genes in pink. Sequences corresponding to S ORFs (aa 1-1,273) from Wuhan-Hu-1, Delta or B.1.1.529 strains were codon-optimized for expression in humans, the furin cleavage site “RRAR” was removed and replaced by “GSAS” [22], and six proline substitutions were introduced to stabilize the S antigen in the prefusion form [23]. An additional gene encoding the respective S ORF, flanked by a BPIV3 gene start (light grey) and gene end (dark grey) signal sequence, was inserted between the BPIV3 N and P genes [25,26]. (B) Vero cells seeded in 6-well plates were mock-infected or infected with the indicated virus using an MOI of 1 PFU/cell and incubated at 32°C for 48 h . Cell lysates were harvested and analyzed by SDS-PAGE under denaturing and reducing conditions and by western blotting. The SARS-CoV-2 S protein, the BPIV3 N protein and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; included as loading control) were detected using primary antibodies (see Methods) followed by immunostaining with infrared fluorophore labeled secondary antibodies and infrared imaging. Images were acquired and analyzed using Image Studio software (LiCor). The approximate molecular weight in kDa is shown on the left. (C) Timeline of the hamster experiment. Five to six-week-old hamsters in groups of 46 were intranasally immunized with 5 log10 PFU of B/HPIV3 empty vector or B/HPIV3 expressing the S-6P, S-Delta-6P or S-Omicron-6P. (D) On days 3 and 5 post-immunization (pi), five hamsters per group each day were euthanized, and vaccine titers were determined from nasal turbinates (D, left panel) and lungs (D, right panel) by dual-staining immunoplaque assay. The limit of detection (dotted line) is 50 PFU/g of tissue. Geometric mean titers (GMTs) for each group are indicated above x axes. Two-way ANOVA with Sidak post-test; exact p values are indicated for levels of significance p < 0.05. (E) Percentages of S-positive plaques in nasal turbinates on days 5 pi. Due to low level of replication on day 3 pi in nasal turbinatesand on day 3 and 5 pi in lungs, these samples were not investigated. Each animal is represented by a circle and GMTs with geometric standard deviations are shown.
Fig 2.
Mucosal and serum antibody responses in immunized hamsters.
(A) IgG (top and bottom left panels), IgA (top and bottom middle panels) and secretory IgA (sIgA) (top right panel) anti-S mucosal antibody titers were evaluated against Wuhan-Hu-1 S (top panels) or B.1.1.529/Omicron S (bottow panels) by ELISA (IgG) or DELFIA (IgA and sIgA) from nasal washes (NW) collected on day 21 post-immunization (pi, n = 18 hamsters per group picked at random). The fraction of animals with titers above the limit of detection is indicated above x axes. (B-E) On day 24 or 25 pi, serum was collected from n = 36 hamsters per group. (B) The IgG (left panels) and IgA (right panels) anti-S antibody titers were evaluated by ELISA (IgG) or DELFIA (IgA) using purified preparations of S antigen from the Wuhan-Hu-1 strain (top row), B.1.617.2/Delta (middle row) or B.1.1.529/Omicron variants (bottom row). ELISA results using purified RBD antigen preparations from the Wuhan-Hu-1 strain are shown in S2 Fig. The limit of detection of reciprocal antibody titers (dotted line) is 1 log10 (A) or 2 (B) log10. (C) Sera collected on day 24 or 25 pi were also analyzed for SARS-CoV-2 neutralizing antibody titers. Live virus neutralization assays were performed using the WA1/2020 strain or B.1.617.2/Delta or BA.1/Omicron variants. Titers were expressed as 50% neutralizing doses. (A-C) Non-parametric Kruskal Wallis test with Dunn post hoc test (Fig 2A and C) and One-way ANOVA with Tukey or Sidak post-test for other data sets; exact p values are indicated for levels of significance p < 0.05. Each hamster is represented by a symbol and medians with interquartile ranges are shown. (B-C) GMTs are indicated above x axes. (D-E) Binding inhibition of soluble ACE2 protein to SARS-CoV-2 S proteins from 20 different variants by serum antibodies from immunized hamsters. Data are represented as a heatmap with the median percent inhibition (from n = 36 hamsters per group) of ACE2 binding relative to a non-serum control indicated for each variant (D), or as a radar plot, with each segment representing a variant, and percent inhibition indicated by concentric circles (E).
Fig 3.
Weight change and lung inflammatory responses of immunized hamsters upon SARS-CoV-2 challenge.
On day 32 pi, the 36 remaining hamsters per immunized group were sub-divided into three subgroups of 12 hamsters each. Each subgroup was challenged with 4.5 log10 TCID50 per animal of SARS-CoV-2 WA1/2020, B.1.617.2/Delta or BA.1/Omicron (see Fig 1C for timeline). (A) Body weights were monitored daily from day 0 to 12 post-challenge (pc). Data are shown as mean percent body weight relative to the day 0 weight (n = 12 hamsters/subgroup from day 0 to 3, n = 6 hamsters/subgroup from day 4 to 12 except for B/HPIV3-immunized and WA1-challenged subgroup with 4 or 5 animals from day 8 to 12). Mixed-effects analysis with Dunnett post-test; exact p values are indicated for levels of significance p < 0.05. (B) Expression of inflammatory/antiviral genes in lungs on day 3 pc. On day 3 pc, six hamsters per subgroup were euthanized and lungs were harvested and homogenized. Total RNA was extracted from lung homogenates and the expression of 10 inflammatory/antiviral genes was evaluated by RT-qPCR using TaqMan assays [25]. Results were analyzed using ΔΔCt method and normalized to beta actin. Relative expression was expressed as log2 fold change over the expression level in five unimmunized, unchallenged hamsters [25] and represented as heatmaps with one heatmap per challenge virus. Non-detected genes are crossed.
Fig 4.
Protection of immunized hamsters from SARS-CoV-2 challenge virus replication.
On day 3 pc, 6 of 12 hamsters per subgroup were euthanized and nasal turbinates and lungs were harvested, and homogenized. Aliquots were used to evaluate SARS-CoV-2 challenge virus replication by RT-qPCR (A) or viral culture (B). (A) Total RNA was extracted from aliquots of lung homogenates and SARS-CoV-2 viral loads were determined by RT-qPCR using TaqMan assays to quantify subgenomic E or N mRNA (Limit of detection: 5.1 log10 copies per g, dotted line). (B) Titers of SARS-CoV-2 challenge viruses, WA1/2020, B.1.617.2/Delta or BA.1/Omicron were determined from aliquots of nasal turbinate and lung homogenates and expressed in log10 TCID50 per g of tissue (Limit of detection: 1.5 log10 TCID50 per g, dotted line). Each hamster is represented by a symbol and GMTs with standard deviations are shown. GMTs are also indicated above x axes. Non-parametric Kruskal Wallis test with the Dunn post hoc test; exact p values are indicated for levels of significance p < 0.05.
Fig 5.
Mucosal and serum antibody responses in immunized hamsters upon SARS-CoV-2 challenge.
On day 23 or 24 pc, the six remaining hamsters per subgroup were euthanized, and bronchoalveolar lavage (BAL) and sera were collected (see Fig 1C for timeline of the experiment) for evaluation of the antibody response by ELISA (IgG) or DELFIA (IgA and sIgA) (A-C) or ACE2 binding inhibition assay (D-E). (A) Anti-S IgG (left panel), IgA (middle panel) and sIgA (right panel) antibody titers in BAL (limit of detection: 1 log10, dotted line for IgG and IgA). N = 6 hamsters per subgroup with the exception of n = 5 for B/HPIV3 immunized/WA1/2020 challenged, B/HPIV3/S-6P-immunized/BA.1 challenged, B/HPIV3/S-Delta-6P-immunized/BA.1 challenged, B/HPIV3/S-Omicron-6P-immunized/WA1/2020 challenged and n = 4 for B/HPIV3/S-6P-immunized/Delta challenged. The anti-S sIgA DELFIA titers were determined for hamsters with sufficient volumes of remaining BAL samples, and the animal numbers and fractions of hamsters with detectable levels are indicated on top of the graph. (B-C) Serum anti-S IgG (B) and IgA (C) titers after challenge (left panels) and fold changes (in log10) of post-challenge titers over post-immunization titers (right panels). N = 6 with the exception of n = 5 for B/HPIV3-immunized/WA1/2020 challenged. Purified preparations of S from the Wuhan-Hu-1 strain were used as antigens in the ELISA. Each hamster is represented by a symbol and medians with interquartile ranges are shown. One-way ANOVA with Tukey post-test; exact p values are indicated for levels of significance p < 0.05. (D-E) Binding inhibition of soluble ACE2 protein to SARS-CoV-2 S proteins from 20 different variants by serum antibodies from immunized and challenged hamsters. Data are represented as a heatmap with one heatmap per challenge virus and with the median percent inhibition of ACE2 binding relative to a non-serum control indicated (D) or as radar plots (E). N = 6 with the exception of n = 5 for B/HPIV3-immunized/WA1/2020 challenged.