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. 2023 Dec;12(1):2192821.
doi: 10.1080/22221751.2023.2192821.

Bivalent vaccines effectively protect mice against influenza A and respiratory syncytial viruses

Sathya N Thulasi Raman  1 , Adrian Zetner  2 , Anwar M Hashem  3   4 , Devina Patel  1 , Jianguo Wu  1 , Caroline Gravel  1 , Jun Gao  5 , Wanyue Zhang  1   6 , Annabelle Pfeifle  1   6 , Levi Tamming  1   6 , Karan Parikh  1 , Jingxin Cao  2 , Roger Tam  1 , David Safronetz  2 , Wangxue Chen  7 , Michael J W Johnston  1   8 , Lisheng Wang  6 , Simon Sauve  1 , Michael Rosu-Myles  1   6 , Gary Van Domselaar  2 , Xuguang Li  1   6
Affiliations

Bivalent vaccines effectively protect mice against influenza A and respiratory syncytial viruses

Sathya N Thulasi Raman et al. Emerg Microbes Infect. 2023 Dec.

Abstract

Influenza and Respiratory Syncytial virus (RSV) infections together contribute significantly to the burden of acute lower respiratory tract infections. Despite the disease burden, no approved RSV vaccine is available. While approved vaccines are available for influenza, seasonal vaccination is required to maintain protection. In addition to both being respiratory viruses, they follow a common seasonality, which warrants the necessity for a concerted vaccination approach. Here, we designed bivalent vaccines by utilizing highly conserved sequences, targeting both influenza A and RSV, as either a chimeric antigen or individual antigens separated by a ribosome skipping sequence. These vaccines were found to be effective in protecting the animals from challenge by either virus, with mechanisms of protection being substantially interrogated in this communication.

Keywords: Adenovirus; HA-stem; RSV; RSV T-cell epitopes; influenza; pre-fusion stabilized F.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Recombinant adenovirus constructs and in vitro protein expression. (a) Schematic representation of the recombinant adenovirus (rAd) constructs. All the constructs were designed to express the HA stem domain, which contains the tyrosinase signaling peptide (SP) at the N-terminus of HA1-stem. (b) In vitro protein expression in mouse cell line. TC-1 mouse lung cells were infected with rAds and cell lysates were collected. Protein expression was confirmed using anti-HA2 polyclonal and anti-RSV-F (palivizumab) monoclonal antibodies. The line arrows indicate the HAstem and HAstem-RSVT-cell protein bands detected by the anti-HA2 antibody.
Figure 2.
Figure 2.
Vaccination induced robust production of anti-HA2 and anti-RSV-F serum antibody titers. (a) Influenza HA2-specific circulating IgG, IgG1 and IgG2a antibodies at 3 weeks after boosting are shown (n = 5). Statistical analysis performed using Kruskal Wallis (non-parametric) test. Data shown is mean ± SEM, **p < 0.01. (b) HA2-specific serum IgG2a:IgG1 ratio indicating Th2- or Th1-biased nature of the immune response (n = 5). Dotted line denotes the threshold (y = 1) for determining immune responses as Th1 or Th2 biased. Statistical analysis performed using one-way ANOVA with Tukey's post hoc test. Data shown is mean ± SEM. ns = not significant. (c) RSV F-specific circulating IgG, IgG1 and IgG2a antibodies at 3 weeks after boosting are shown (n = 5). Statistical analysis performed using Kruskal Wallis (non-parametric) test. Data shown is mean ± SEM, **p < 0.01. (d) RSV F-specific serum IgG2a:IgG1 ratio indicating Th2- or Th1-biased nature of the immune response (n = 5). Dotted line denotes the threshold (y = 1) for determining immune responses as Th1 or Th2 biased. Statistical analysis was performed using paired t-test. Data shown is mean ± SEM. ns = not significant.
Figure 2.
Figure 2.
Vaccination induced robust production of anti-HA2 and anti-RSV-F serum antibody titers. (a) Influenza HA2-specific circulating IgG, IgG1 and IgG2a antibodies at 3 weeks after boosting are shown (n = 5). Statistical analysis performed using Kruskal Wallis (non-parametric) test. Data shown is mean ± SEM, **p < 0.01. (b) HA2-specific serum IgG2a:IgG1 ratio indicating Th2- or Th1-biased nature of the immune response (n = 5). Dotted line denotes the threshold (y = 1) for determining immune responses as Th1 or Th2 biased. Statistical analysis performed using one-way ANOVA with Tukey's post hoc test. Data shown is mean ± SEM. ns = not significant. (c) RSV F-specific circulating IgG, IgG1 and IgG2a antibodies at 3 weeks after boosting are shown (n = 5). Statistical analysis performed using Kruskal Wallis (non-parametric) test. Data shown is mean ± SEM, **p < 0.01. (d) RSV F-specific serum IgG2a:IgG1 ratio indicating Th2- or Th1-biased nature of the immune response (n = 5). Dotted line denotes the threshold (y = 1) for determining immune responses as Th1 or Th2 biased. Statistical analysis was performed using paired t-test. Data shown is mean ± SEM. ns = not significant.
Figure 3.
Figure 3.
Serum antibodies possess neutralizing and ADCC effector functions against influenza and RSV. (a,b) Serum collected post-boosting was used to determine the neutralizing ability (a) and ADCC activity (b) against influenza A/Netherlands/602/09 (H1N1). Anti-Influenza A/California/7/09 HA serum (NIBSC code – 14/310) was used as a positive control for the neutralization assay. RSV-A2 neutralizing ability (c) and ADCC activity against RSV-A2 (d) of the mice serum collected post-boosting (n = 5) is shown. (b,d) Fold induction over “no antibody” control is shown. Data shown is mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. (two-wayANOVA with Tukey's post hoc test (b), paired t-test (c), Dunnett's post hoc test (d)).
Figure 4.
Figure 4.
RSV CD4 and CD8 epitope peptide stimulation induces cytokine secretion from immune cells isolated from rAd-HAstem-RSVTcell vaccinated mice. (a) Splenocytes and Lymph node cells were incubated with a pool of six T-cell epitope peptides at 5 µg/ml each (n = 5). A panel of Th1/Th2 secreted cytokines were measured at 24 h of incubation. Data shown is mean ± SEM. (b) Splenocytes and Lymph node cells were incubated with each of the six T-cell epitope peptides (Table 1) at 5 µg/ml (n = 5). A panel of Th1/Th2 secreted cytokines were measured at 24 h of incubation. Data shown is mean ± SEM. Statistical data corresponds to comparison of vaccinated and control mice. (c) Splenocytes from vaccinated animals were stimulated with a pool of the three CD8 epitope peptides for 5 days and were co-incubated with peptide-pulsed P815 target cells for 4 h (n = 5). Peptide-specific target cell lysis was calculated based on the amount of LDH released in the media. Data shown is mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (t-tests (a,b,c))
Figure 4.
Figure 4.
RSV CD4 and CD8 epitope peptide stimulation induces cytokine secretion from immune cells isolated from rAd-HAstem-RSVTcell vaccinated mice. (a) Splenocytes and Lymph node cells were incubated with a pool of six T-cell epitope peptides at 5 µg/ml each (n = 5). A panel of Th1/Th2 secreted cytokines were measured at 24 h of incubation. Data shown is mean ± SEM. (b) Splenocytes and Lymph node cells were incubated with each of the six T-cell epitope peptides (Table 1) at 5 µg/ml (n = 5). A panel of Th1/Th2 secreted cytokines were measured at 24 h of incubation. Data shown is mean ± SEM. Statistical data corresponds to comparison of vaccinated and control mice. (c) Splenocytes from vaccinated animals were stimulated with a pool of the three CD8 epitope peptides for 5 days and were co-incubated with peptide-pulsed P815 target cells for 4 h (n = 5). Peptide-specific target cell lysis was calculated based on the amount of LDH released in the media. Data shown is mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (t-tests (a,b,c))
Figure 5.
Figure 5.
Intranasal immunization with the vaccine constructs provide significant protection against influenza and RSV challenge. (a) Schematic diagram of the immunization, influenza A challenge and necropsy timeline at a challenge dose of 1250 PFU/mouse. (b–d) BALB/c mice were vaccinated intranasally in a prime/boost regimen, 4-weeks apart, with the rAd vaccines and challenged intranasally with 1250 PFU/mouse of influenza A. (b) Survival (n = 7 or 8) is shown. Median survival time since day of challenge – Ad-empty: 7 days, all vaccinated groups: > 14 days. *p < 0.05, **p < 0.01, ***p < 0.001, log-rank test. (c) Weight loss data is shown. Data shown is mean ± SEM. (d) Virus titre from infected lungs and nose collected 5 days post-challenge were determine by plaque assay (n = 4 or 5). Data shown is mean ± SEM. The dotted line denotes the limit of detection (LOD) of the assay. *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVA (Tukey's post hoc test) (c), one-way ANOVA (Dunnet's post hoc test) for lung viral titers and Kruskal-Wallis (non-parametric) test for the Nose viral titers (d). (e) Schematic diagram of the immunization, RSV-A2 challenge and necropsy timeline. (f) BALB/c mice were vaccinated intranasally in a prime/boost regimen, 4-weeks apart, with the rAd vaccines and challenged intranasally with RSV-A2. Virus titre from infected lungs collected 4 days post-challenge were determine by plaque assay (n = 5). Data shown is mean ± SEM. ***p < 0.001.one-way ANOVA (Dunnet's post hoc test).
Figure 6.
Figure 6.
Pathological scoring of lung tissue following virus challenge. (a) Average pathological score of lung tissue from mice 5 days post-challenge with influenza A. (b) Representative images of H&E stained mice lungs post challenge with influenza at 20× magnification. Long arrows point to perivascular cuffing, short arrows point to epithelial cell hyperplasia and arrowheads point to necrotic epithelial cells. Length of the scale bar is 50 µm. (c) Average pathological score of lung tissue from mice, 4 days post-challenge with RSV-A2. (d) Representative images of H&E stained mice lungs post challenge with RSV at 20× magnification. Long arrows point to perivascular cuffing. Length of the scale bar is 50 µm. Data shown is mean ± SEM, *p < 0.05 (Kruskal Wallis (non-parametric) test).
Figure 6.
Figure 6.
Pathological scoring of lung tissue following virus challenge. (a) Average pathological score of lung tissue from mice 5 days post-challenge with influenza A. (b) Representative images of H&E stained mice lungs post challenge with influenza at 20× magnification. Long arrows point to perivascular cuffing, short arrows point to epithelial cell hyperplasia and arrowheads point to necrotic epithelial cells. Length of the scale bar is 50 µm. (c) Average pathological score of lung tissue from mice, 4 days post-challenge with RSV-A2. (d) Representative images of H&E stained mice lungs post challenge with RSV at 20× magnification. Long arrows point to perivascular cuffing. Length of the scale bar is 50 µm. Data shown is mean ± SEM, *p < 0.05 (Kruskal Wallis (non-parametric) test).
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Grants and funding

This work was supported by Health Canada [grant number 2021].

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