A Universal Influenza Vaccine Can Lead to Disease Exacerbation or Viral Control Depending on Delivery Strategies

Abstract

The development of influenza A virus (IAV) vaccines, which elicits cross-strain immunity against seasonal and pandemic viruses is a major public health goal. As pigs are susceptible to human, avian, and swine-adapted IAV, they would be key targets of so called universal IAV vaccines, for reducing both the zoonotic risk and the economic burden in the swine industry. They also are relevant preclinical models. However, vaccination with conserved IAV antigens (AGs) in pigs was reported to elicit disease exacerbation. In this study, we assessed whether delivery strategies, i.e., dendritic cell (DC) targeting by the intradermal (ID) or intramuscular (IM) routes, impact on the outcome of the vaccination with three conserved IAV AGs (M2e, NP, and HA2) in pigs. The AGs were addressed to CD11c by non-covalent binding to biotinylated anti-CD11c monoclonal antibody. The CD11c-targeted AGs given by the ID route exacerbated disease. Conversely, CD11c-targeted NP injected by the IM route promoted T cell response compared to non-targeted NP. Furthermore, the conserved IAV AGs injected by the IM route, independently of DC targeting, induced both a reduction of viral shedding and a broader IgG response as compared to the ID route. Our findings highlight in a relevant animal species that the route of vaccine delivery impacts on the protection induced by conserved IAV AGs and on vaccine adverse effects. Finally, our results indicate that HA2 stands as the most promising conserved IAV AG for universal vaccine development.

Keywords: dendritic cells; human; influenza; routes of administration; swine; vaccine.

Figure 1

SDS-PAGE migration of tetrameric SA-HA2, SA-M2e, SA-NP, and HA2-SA-M2a proteins. (A) Schematic depiction of the various pET28b-AG fused to streptavidin (SA–AG) expression constructs. (B) SDS-PAGE analysis of the purified soluble and heat-denatured (H) monomers of SA–AG fusion proteins of their native tetramers formed upon dialysis against 50 mM ammonium carbonate pH 8.9. The proteins were separated by Tris–Tricin SDS-PAGE and were visualized by Coomassie blue staining. The tetramers were disrupted by sample heating at 100°C for 5 min (H).

Figure 2

Binding of CD11c-VC on pig alveolar macrophages and skin dendritic cell (DC). (A) Binding of CD11c-VC on pig alveolar macrophages. Left panel: broncho-alveolar lavage (BAL) cells were incubated with biot-CD11c monoclonal antibodies (mAb) or biot-isotype control (ISC) followed by Alexa 647-conjugated streptavidin; SSC^high FCS^high cells (mostly macrophages) were gated and the fluorescent signals were analyzed (biot-CD11c mAb, plain line versus biot-ISC mAb, filled gray). Right panel: BAL cells were incubated with preformed CD11c-VC and ISC-VCn followed by anti-NP rabbit IgG + anti-MHC class II mAb, and Alexa 647-conjugated anti-rabbit + Alexa 488-conjugated anti-mouse IgG2a secondary Ab. After gating the MHC2 class II⁺ cells (alveolar macrophages), the anti-NP fluorescent signals obtained with biot-CD11c (plain line) or biot-ISC mAb (filled gray) are shown. (B) Binding of CD11c-VC on pig skin-migrated DC. Migrated cells from skin explants were collected and incubated as in (A). Live (DAPI negative) and MHC class II positive cells were gated and the anti-NP fluorescent signals obtained with biot-CD11c (plain line) or biot-ISC mAb (filled gray) are depicted.

Figure 3

Ab and T cell responses induced by vaccicomplexes (VCs) upon intradermal (ID) inoculation with and without CpG. Pigs from experiment 1 were immunized intradermally with CD11c-VC-NP and CD11c-VC-HA2-M2e (designated as CD11c on the figure), isotype control (ISC)-VC-NP and ISC-VC-HA2-M2e (designated as ISC), and uncomplexed AG fused to streptavidin (designated as AG) at 0 and 30 dpv, with or without CpG (500 µg). The anti-NP, HA2, and M2e IgG titers are shown in (A–C), respectively, at 0 and 55 dpv. In (D), the T cell response was assessed by restimulating spleen cells (at 55 dpv) with recombinant NP for 3 days and measuring released IFNγ by ELISA. As recombinant standard was not used, only OD are provided. Each dot corresponds to individual pig values. Means and SEM are shown. Statistical significance between the Ab response at 55 dpv versus d0 was calculated with paired bilateral t-tests and significance was always found in the case of anti-NP (p < 0.02), and anti-HA2 IgG (p < 0.05). Comparison between vaccinated groups at 55 dpv was done with a one-way ANOVA and a Newman–Keuls multiple comparison test, and significance is shown (**p < 0.01, *p < 0.05).

Figure 4

Ab and T cell responses induced by vaccicomplexes (VCs) upon intradermal (ID) and intramuscular (IM) inoculation without adjuvant. Pigs from experiment 2 were immunized either by ID or IM routes, with CD11c-VC-NP + CD11c-VC-HA2 + CD11c-VC-M2e (designated as CD11c), or isotype control (ISC)-VC-NP + ISC-VC-HA2 + ISC-VC-M2e (designated as ISC) at 0 and 50 dpv. The anti-NP, HA2, and M2e IgG titers are shown in (A–C), respectively, at 0 and 75 dpv. In (D), the T cell response was assessed by restimulating fresh PBMCs collected at 70 dpv with recombinant NP for 3 days and measuring released IFNγ by ELISA. As recombinant standard was not used, only DO are provided. Each dot corresponds to individual pig values. Means and SEM are shown. Comparison between vaccinated groups and the control non-vaccinated group was done with a one-way ANOVA and a Dunnett’s comparison test, and significance is shown (***p < 0.001, **p < 0.01, *p < 0.05).

Figure 5

Clinical symptoms (A) and viral detection (B) in nasal swabs of pigs immunized with vaccicomplexes. Pigs from experiment 2 (see Figure 4) were challenged by the intratracheal route with pandemic (H1N1) 2009. (A) Clinical symptoms, i.e., nasal discharge or conjunctivitis, monitored for each pig from 2 to 8 dpc are reported with a color code, with one symptom in orange, two symptoms in red. Pigs are designated by their breeding number and presented in their assigned groups under the gray tag. (B) Viral detection in nasal swab was done daily for each pig from 3 to 8 dpc using plaque assay. PFU/ml superiors to 10⁵, 10⁴, 10³, and 10² are represented by a color code from dark red to yellow, respectively. A white rectangle corresponds to absence of detected virus.

Figure 6

Principal component analysis (PCA) analysis of the viral shedding and immune responses of vaccicomplexes (VCs)-vaccinated pigs. (A) PCA plot of the responses of pigs is depicted with each pig represented as a dot in a specific color according to its group assignment: CD11c-VC intradermal (ID-CD11c, blue), isotype control (ISC)-VC intradermal (ID-ISC, red), CD11c-VC intramuscular (IM-CD11c, green), ISC-VC intramuscular (IM-ISC, black). PC1 explained 28.9% of the total variation between pigs and PC2 explained a further 17.71% of the variation. (B) PCA loading for each individual input variable: viral shedding at 3, 4, 5, 6 dpc (VS-D3, VS-D4, VS-D5, VS-D6), global clinical symptoms over 2–8 dpv (Clinic), T cell response at 70 dpv (T-cell), anti-HA2, anti-M2e, anti-NP Ab response on the day of the challenge (HA2-D0, M2e-D0, NP-D0) and at 5 dpc (HA2-D5, M2e-D5, NP-D5). Note that the day of challenge (D0 in Figure 6) corresponds to 75 dpv in Figure 4.