Prime-pull vaccination with a plant-derived virus-like particle influenza vaccine elicits a broad immune response and protects aged mice from death and frailty after challenge

Abstract

Background: Administered intramuscularly (IM), plant-derived, virus-like-particle (VLP) vaccines based on the influenza hemagglutinin (HA) protein elicit both humoral and cellular responses that can protect aged mice from lethal challenge. Unlike split virus vaccines, VLPs can be administered by different routes including intranasally (IN). We evaluated novel vaccine strategies such as prime-pull (IM boosted by IN) and multi-modality vaccination (IM and IN given simultaneously). We wished to determine if these approaches would provide better quality protection in old mice after less severe (borderline-lethal) challenge (ie: immunogenicity, frailty and survival).

Results: Survival rates were similar in all vaccinated groups. Antibody responses were modest in all groups but tended to be higher in VLP groups compared to inactivated influenza vaccine (IIV) recipients. All VLP groups had higher splenocyte T cell responses than the split virus group. Lung homogenate chemokine/cytokine levels and virus loads were lower in the VLP groups compared to IIV recipients 3 days after challenge (p < 0.05 for viral load vs all VLP groups combined). The VLP-vaccinated groups also had less weight loss and recovered more rapidly than the IIV recipients. There was limited evidence of an immunologic or survival advantage with IN delivery of the VLP vaccine.

Conclusion: Compared to IIV, the plant-derived VLP vaccine induced a broader immune response in aged mice (cellular and humoral) using either traditional (IM/IM) or novel schedules (multi-modality, prime-pull).

Keywords: Aged mouse model; Frailty; Influenza vaccines; Multi-modality; Prime-pull; Virus-like particles.

Fig. 1

Timeline for vaccine administration. Female BALB/c mice (18–22 months of age) were vaccinated twice on day 0 (d0) and day 21 (d21) with plant-derived H1-VLP vaccine, inactivated H1N1 split vaccine or PBS. Three groups of animals received the VLP vaccine: i) two 3 μg doses intramuscularly (IM/IM) ii) a first 3 μg dose IM boosted at d21 by a 3 μg dose intranasally (IM/IN: Prime-Pull group) or iii) two doses of 1.5 μg IM plus 1.5 μg IN (IM + IN: Multi-modality group). The comparator group received two 3 μg doses of a split inactivated influenza vaccine. Peripheral blood was collected at d0 (pre-vaccination) and at d21 (data not shown) and d42 after the first dose of vaccine. Spleens and lungs were harvested from individual animals. The remaining mice (typically 6–8 mice/group) were scored for frailty on d40–42 then challenged with a sub-lethal dose of wild-type H1N1 A/California/07/2009 virus (525 TCID₅₀ in 50 μL) by IN instillation (25 μL/nare). Weight loss was monitored daily for up to 28 days. At d45 or 3 days post-infection (dpi), 3–5 mice/group were sacrificed (isoflurane/CO₂) and serum (cardiac puncture) and lungs were collected. At d67 (25 ± 4 dpi), surviving mice were scored for frailty and sacrificed to collect serum and lungs

Fig. 2

Survival after A/California/07/2009 H1N1 challenge. Aged BALB/c female mice (18–22 months of age) were immunized twice with H1-VLP vaccine or inactivated split vaccine. Six weeks after vaccination, mice were challenged with a sub-lethal dose of A/California/07/2009 H1N1 and were closely monitored for weight loss. Mice were euthanized if they lost > 20% of their initial weight A log-rank (Mantel-Cox) test was used to compare survival curves with the PBS control group (** p < 0.01, * p < 0.05). This graph is representative of 5–10 mice/group from two separate studies

Fig. 3

Antibody responses after two vaccinations towards H1N1 A/California/07/2009. Aged BALB/c female mice (18–22 months of age) were immunized twice with H1-VLP or inactivated split vaccine. Six weeks post-vaccination, the humoral response to the H1 of A/California/07/2009 H1N1 was analyzed in sera from individual mice by hemagglutination inhibition (HAI: 8–10 animals/group) (a), micronuetralization (MN: 6–8 animals/group) (b) and ELISA (c: 8–10 animals/group). The dotted line in a represents an HAI titre of 1:40, which is considered protective in humans. Error bars indicate 95% CI. For statistical analysis, one-way ANOVA was used on log transformed values (*** p < 0.001, ** p < 0.01 * p < 0.05). These data represent 2 independent studies

Fig. 4

Splenocyte T cells expressing cytokines in response to H1 re-stimulation ex-vivo. Splenocytes were collected 3 weeks post-boost from aged female BALB/c mice (18–22 months of age). Percent of splenocytes a CD4⁺ T cells and b CD8⁺ T cells expressing 2 or more cytokines (polyfunctional) or single cytokines (IFNγ, IL-2 or TNF⍺). For statistical analysis, two-way ANOVA was performed followed by Tukey’s multiple comparison test (**** p < 0.0001, *** p < 0.001, * p < 0.05) compared to the split vaccine. a and b are representative data from 6 to 8 mice/group from two studies

Fig. 5

Lung viral loads and weight loss after challenge. Aged BALB/c mice (18–22 months) lungs were collected at 3 days post-infection after sub-lethal challenge with H1N1 A/California/07/2009. Three days post-infection a lung viral loads were measured and throughout the infection mice were closely monitored for b weight loss. a is representative of 3–5 mice/group from two studies and b are representative of 5–10 mice/group combined from two studies. For statistical analysis, a One-way ANOVA was performed on the log10 values of the viral titres and the Tukey’s multiple comparison test was performed. For b Two-way ANOVA followed by the Tukey’s multiple comparison test (**** p < 0.0001, *** p 0.001, ** p < 0.01, * p < 0.05) (see Additional file 1: Table S2). Error bars represent the standard error of the mean

Fig. 6

Cytokine and chemokine levels in lung homogenates 3 days after challenge. Six weeks post-vaccination, female BALB/c mice (18–22 months) were challenged with A/California/07/2009 H1N1 and lungs were collected and homogenized 3 days post-infection to measure cytokines/chemokines by multiplex ELISA for the following cytokines: a IL-1α, IL-6, MCP-1, MIP-1α, RANTES and b IL-1β, IL-2, IL-3, IL-4, IL-5, IL-10, IL-12, IL-17, IFNγ, TNFα, GM-CSF. For statistical analysis, one-way ANOVA followed by Tukey’s multiple comparison test (**p < 0.01, * p < 0.05). Data represent 4–7 mice/group from one study. Error bars represent the standard error of the mean

Fig. 7

Changes in clinical frailty index after challenge. At day 0 (post-vaccination but pre-challenge) and day 25 post-infection, clinical frailty indices were measured. The percent difference was calculated for each mouse that survived (25 dpi-0 dpi*100). A proportion of those with ≥3% change in each group was also calculated. Assigned frailty changes due to death are outlined in black. For statistical analysis, one-way ANOVA followed by Tukey’s multiple comparison test was used to compared to groups. Data is representative of 6–8 mice/group from 2 studies