Sequential immunization with chimeric hemagglutinin ΔNS1 attenuated influenza vaccines induces broad humoral and cellular immunity

Abstract

Influenza viruses pose a threat to public health as evidenced by severe morbidity and mortality in humans on a yearly basis. Given the constant changes in the viral glycoproteins owing to antigenic drift, seasonal influenza vaccines need to be updated periodically and effectiveness often drops due to mismatches between vaccine and circulating strains. In addition, seasonal influenza vaccines are not protective against antigenically shifted influenza viruses with pandemic potential. Here, we have developed a highly immunogenic vaccination regimen based on live-attenuated influenza vaccines (LAIVs) comprised of an attenuated virus backbone lacking non-structural protein 1 (ΔNS1), the primary host interferon antagonist of influenza viruses, with chimeric hemagglutinins (cHA) composed of exotic avian head domains with a highly conserved stalk domain, to redirect the humoral response towards the HA stalk. In this study, we showed that cHA-LAIV vaccines induce robust serum and mucosal responses against group 1 stalk and confer antibody-dependent cell cytotoxicity activity. Mice that intranasally received cH8/1-ΔNS1 followed by a cH11/1-ΔNS1 heterologous booster had robust humoral responses for influenza A virus group 1 HAs and were protected from seasonal H1N1 influenza virus and heterologous highly pathogenic avian H5N1 lethal challenges. When compared with mice immunized with the standard of care or cold-adapted cHA-LAIV, cHA-ΔNS1 immunized mice had robust antigen-specific CD8⁺ T-cell responses which also correlated with markedly reduced lung pathology post-challenge. These observations support the development of a trivalent universal influenza vaccine for the protection against group 1 and group 2 influenza A viruses and influenza B viruses.

Fig. 1. Characterization of cHA-LAIV vaccine strains in vitro.

a The final purified viral stocks were used to assess the comparative replication competence in Vero-CCL81 cells at a multiplicity of infection (MOI – 0.001) in triplicate and all viral supernatants were assessed by plaque assay. b Vero cells were infected at an MOI – 5 for 16 h for a single replication cycle and cells were stained using the broadly neutralizing antibody CR9114 for flow cytometric analysis. Infection percentages are shown for each virus. c cHA-LAIVs were plaqued in MDCK and MDCK-NS1 (only for ΔNS1 viruses) at 33 or 37 °C for 48 h. Plaques were immunostained using HT103 mAb (anti-NP) to assess temperature sensitive and attenuated phenotypes. d Amino acid changes noted in the HAs of cHA-ΔNS1 after passaging 10 times after virus rescue. Statistics were done using one way-ANOVA conducting multiple comparison against cH8/1-ΔNS1. **P < 0.083, ***P < 0.0009 and ****P < 0.0001. Data are shown as mean ± SE.

Fig. 2. cHA-LAIV study design and sample collection.

a Animals were randomly assigned into seven groups (n = 35). Groups 1-5 were intranasally infected with a sub-lethal dose of purified cH9/1-IBV (2 × 10⁵ PFU). Mice were immunized in 4-week intervals. Group 1 received cH8/1-N1-ΔNS1 and then cH11/1-N1-ΔNS1, group 2 received the vaccine doses in reverse order (10⁵ PFU per dose). Groups 3 and 4 followed the regimens of groups 1 and 2 except that the strains had a cold-adapted temperature-sensitive LAIV backbone (with a full length, functional NS1). Group 5 and 6 were mock immunized with identical volumes of sterile 1x PBS. Group 7 was intramuscularly given 50 µl of non-adjuvanted QIV per dose (~1.5 µg per dose). We used Group 7 as a “standard-of-care” vaccine comparator. Although this group does not include a cH9/1-IBV, previous experiments demonstrated that even in the presence of priming, QIV vaccination did not confer good protection against heterologous challenges. Animals were intranasally challenged 4 weeks post final-boost either with IVR-180 (BSL-2; 100x LD₅₀) or influenza A/Vietnam/1203/04 virus (BSL-3 + ; 20x LD₅₀) or 13 weeks post final-boost for a separate BSL-3+ challenge (1000x LD₅₀). b Animals were bled (submandibular), and nasal washes were taken at D0 (naïve) and D98 animals. Mice were bled between immunization doses at approximately 28-day intervals up until D98 since the first dose. c Sample collection for BSL2 100 LD₅₀ challenge included submandibular bleeds at D1, D5 and D10 (n = 5) for pooled circulating tetramer specific CD8⁺ T-cell analysis. Lungs (left lobes) and nasal turbinates were harvested on D3 and D5 for viral titration. Right lobes of lungs on D3 were used for analysis of tissue-resident memory CD8⁺ T-cell analysis and on D5 for histopathology. Spleens were harvested on D5 for enzyme-linked immunosorbent spot (ELISpot) and intracellular staining assays. For BSL-3+ studies, whole-lung and nasal turbinates were harvested on D3 and D5 for viral titration. Image was created using BioRender.

Fig. 3. cHA-LAIV vaccines induce robust serum and mucosal responses against group 1 stalk and confer ADCC reporter assay activity.

Longitudinal area-under-the-curve values (AUCs) were calculated using endpoint titers from serum/nasal washes derived from mice before, between and after immunization. a Mucosal total IgA titers depicted as AUC values for group 1 HA stalk derived from endpoint titers of (n = 3) from animal nasal washes (undiluted starting dilution) on D98. b Longitudinal group 1 HA stalk total IgG titers from pooled sera. c Individual values of group 1 HA stalk total IgG titers from serum on D98. d ADCC reporter assay activity from serially diluted serum samples (1:20 starting dilutions) in which endpoint titers were used to calculate area under the curve values (AUCs) plotted from 3 sets of pooled animal serum from each respective group. In (a), significance established by comparisons against the group with the highest values (Group 1) while all other panels had comparisons to QIV standard of care group (Group 7) using one way-ANOVA for multiple comparisons corrected and adjusted as per Dunnett’s correction. ****P- < 0.0001, ***P-0.001, **P-0.002, *P-0.02. Data are shown as mean ± SEM. Limits of detection (LOD) is shown in dotted lines. Upper dotted line indicates HA-Stalk Prime only (Group 5) mean.

Fig. 4. cHA-LAIV vaccines induce broad group specific HA and NA total IgG serum responses.

Longitudinal area under the curve values (AUCs) were calculated using endpoint titers from serum derived from mice before, between and after immunization (A-C top panels). Respective individual AUC values on D98 (A-C bottom panels) are also shown. a Total serum IgG responses for full-length H2. b Total serum IgG responses for full-length H9. c Total serum IgG responses for full-length H18. d Total serum IgG responses for full-length H3 (group 2 HA). e Longitudinal serum IgG responses against N1 NA and individual values on D98 for N1-NA responses (right panel). Significance was calculated compared to the QIV standard of care group (Group 7) using one way-ANOVA for multiple comparisons corrected and adjusted as per Dunnett’s correction. ****P- < 0.0001, ***P-0.0001, **P-0.001, *P-0.01. Data are shown as mean ± SEM. Limits of detection (LOD) is shown in dotted lines. Upper dotted line indicates HA-Stalk Prime only (Group 5) mean.

Fig. 5. cHA-LAIV vaccination protects mice from a lethal high-dose seasonal influenza challenge and confers superior upper respiratory tract protection.

Four weeks post-final-boost, mice were challenged with a QIV-matched IVR-180 H1N1 virus using a lethal dose of 100x LD₅₀. a Morbidity was assessed by monitoring weight loss. b Survival of challenged animals. c Viral titers in lungs assessed by plaque assays on day 3 and day 5 post-infection. d Viral titers in nasal turbinates on day 3 and 5 post-infection. Each dot represents one animal. Data are shown as mean ± SEM. Statistical significance was compared to QIV standard of care group (Group 7) using 2 way-ANOVA with Dunnett multiple comparisons. ****P- < 0.0001, ***P-0.0001, **P-0.001, *P-0.01. Limits of detection (LOD) is shown in dotted lines.

Fig. 6. cHA-ΔNS1-LAIV vaccination induces potent antigen-specific cytotoxic CD8 T-cell responses during virus challenge.

Four weeks post-final-boost, mice (n = 5 per group) were challenged with a QIV-matched IVR-180 using a lethal dose of 100x LD₅₀. a A representative image of the gating strategy used to probe NP-tetramer+ CD8⁺ T-cell responses of pooled blood derived from mice on D1, D5, and D10 post-challenge. (The same animals were followed throughout the study). The following markers were utilized for the gating strategy; CD3e – T cell marker, CD8a – CD8 T cell marker. b Amino-acid sequence of the NP tetramer used in this study corresponds to all the NPs of the viruses used in this study. c Longitudinal assessment of IAV-NP⁺CD8⁺ circulating T-cells expressed as a percentage of total CD8⁺ T-cells (left) and percentage of total lymphocytes (right).

Fig. 7. cHA-ΔNS1-LAIV vaccination activates tissue-resident memory (TRM) IAV-NP⁺CD8⁺ T-cells on day 3 post-infection.

Four weeks post-final-boost, mice were challenged with a QIV matched IVR-180 using a lethal dose of 100 LD₅₀. a A representative image of the gating strategy used to probe lung resident NP-tetramer+ CD8⁺ T-cell responses of animals on day 3 post-infection. The following markers were utilized for the gating strategy; CD3e—T cell marker, CD8a—CD8 T cell marker, CD69—Activation marker, CD44—Memory marker, CD103—Tissue resident marker (absent in CD4+) b Absolute count of TRM IAV-NP⁺CD8⁺ T-cells 3 days post infection. Each dot is one animal and data are shown as mean ± SEM Statistical significance was compared to QIV standard of care group (Group 7) using one-way ANOVA using Dunnett’s correction. **** P- <0.0001.

Fig. 8. ELISpot assays for IFNγ⁺CD8⁺ T-cells from splenocytes indicate robust T-cell responses against viral NP and HA stalk, induced by cHA-LAIV vaccinations.

Four weeks post-final-boost, mice were challenged with a QIV-matched IVR-180 using a lethal dose of 100x LD_50. On day 5 post-infection, spleens were harvested and processed for ELISpot assays. Splenocytes (10⁵ cells) were stimulated with indicated CD8 restricted peptides for 16 h. a HA stalk-peptide stimulated. b NP peptide stimulated and c. RSV-F peptide stimulated (irrelevant peptide). Each dot represents one animal. Side panels indicate representative ELISpot images form each animal. Data present as mean ± SEM. Statistical significance was compared to QIV standard of care group (Group 7) using one-way ANOVA using Dunnett’s-correction. ****P- <0.0001, ***P-0.0002, **P-0.0011, *P-0.01. Limits of detection (LOD) are shown in dotted lines.

Fig. 9. cHA-ΔNS1-LAIV vaccination protects mice from severe bronchointerstitial histopathology after seasonal virus challenge.

Four weeks post-final-boost, mice were challenged with a QIV-matched IVR-180 using a lethal dose of 100 LD₅₀. On day 5 post infection left lung lobes were fixed in formalin and were processed for H&E staining and IHC. Lungs were scored by a blinded independent veterinary pathologist to assess pathology features. a Representative images from an animal derived from each group are shown as H&E staining (top panels) and NP IHC immunostaining (bottom panels). Peribronchiolar (black hashed outlines) and perivascular inflammation were observed to variable degrees in all influenza virus-inoculated animals regardless of vaccination group. Normal naïve lungs are represented for comparison. Scale bar = 100 microns. All images acquired at 200x total magnification. b Scoring for IHC (α-NP), c scoring for interstitial pathology, d. scoring for airway epithelial integrity and e. cumulative scores based on the assessed parameters. Scoring for individual parameters range from 0–5: 0 no pathology and 5 being severe pathology. Each dot is derived from a single animal and data is shown as mean ± SEM.

Fig. 10. cHA-LAIV vaccination protects mice from a high dose heterologous highly pathogenic avian influenza challenge and confers superior upper respiratory tract protection.

Foue weeks post final-boost, mice were challenged with an A/VN/1203/04/H5N1 virus using a lethal dose of 20x LD₅₀.Thirteen weeks post final-boost the mice were challenged with high lethal dose 1000x LD₅₀ challenge. a Morbidity (left) and mortality (right) were assessed by monitoring weight loss (20x LD₅₀). b Viral titers in lungs (left) and nasal turbinates (right) on day 3 and 5 post-infection of challenged animals (20x LD₅₀). c Morbidity (left) and mortality (right) were assessed by monitoring weight loss (1000x LD₅₀). d Viral titers in lungs (left) and nasal turbinates (right) on day 3 and 5 post-infection of challenged animals (1000x LD₅₀). Each dot represents one animal. Data is shown as mean ± SEM. Statistical significance was compared to QIV-Standard of care group (Group 7) using one-way ANOVA using Dunnett’s-correction. ****P-<0.0001, ***P-0.0002, **P-0.0011, *P-0.02. Limits of detection (LOD) are shown in dotted lines.