Self-Amplifying Pestivirus Replicon RNA Encoding Influenza Virus Nucleoprotein and Hemagglutinin Promote Humoral and Cellular Immune Responses in Pigs

Figures

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  Figure 1. Prime-boost protocol for i.d. pig immunization. (A) Schematic representation of the CSFV genome (parent virus genome) employed for generating the RepRNA constructs: the deletion of the Erns coding sequence (ΔErns) and the inserted GOI (Rep-NP, Rep-HA) are shown. (B) Illustration of RepRNA functional assay by confocal microscopy, adapted from the infection center assay (ICA) reported elsewhere (25). In this example, SK-6 cells were electroporated with Rep-NP, after what we performed a tenfold serial dilution of the transfected cells (corresponding to 10-1 µg RepRNA and 8 x 105 cells per 100 µl in the first dilution to 10-4 µg RepRNA and 800 cells per 100 µl in the 4th dilution). 100 µl of each dilution containing electroporated cells were transferred to the corresponding Lab-Tek® wells and let to adhere to 3 x 105 pre-seeded SK-6 cells for 4–6 h in the 37°C incubator in MEM/glutamax/7%v/v-horse-serum. Then, the medium was replaced, and cells were cultured for 48 h at 37°C. The staining protocol involved fixation, permeabilisation and labelling with antibody against NP (green) and E2 (red); cell surfaces were stained with WGA-Alexa633 (gray). (C) Illustration of the prime-boost…

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  Figure 2. Virus replicon particle (VRP) and NGA deliver RepRNA for translation in vivo. (A, B) Mice and rabbits were injected subcutaneously at 0, 14, and 28 days. Rep-HA and Rep-NP were delivered in NGA or VRP; all vaccines were adjuvanted with c-di-AMP adjuvant. Serum samples were assessed for anti-HA and anti-NP antibodies by ELISA and titers estimated at the times shown. (C) Adoptive transfer studies in mice have been performed in order to investigate the potential of the polyethylenimine (PEI) formulations to stimulate cellular immune responses. In brief, 24 h prior vaccination of wild type mice, CFSE labeled Thy1.1 CD8+ T cells of antigen-specific TCR transgenic mice were injected intravenously. 7 days later, cervical LNs of transplanted mice were collected and the proliferative capacity of the CFSE labeled Thy1.1 CD8+ T cells was analyzed by flow cytometry. Cell number as well as the number of cell divisions correlate with both strength of the stimulated cellular response and vaccine delivery efficacy. The number of division cycles (D1-D3) of proliferating OT-I CD8+ T cells were determined by flow cytometry (FCM) using the FlowJo software. For each group, tissues from five mice were…

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  Figure 3. Virus replicon particle (VRP) immunization induces specific immune cell proliferation in the draining lymph nodes. (A) The gating strategy used to quantify B and T cell proliferation, based on the expression of CD3, CD4, CD8, CD21, IgM, and CellTrace™ markers, is shown. (B) On the left side is shown a representative histogram, where FCSlow and FSChigh gates can be distinguished. Then, CD4+ T cells (CD3+CD21-IgM-CD4+CD8-), CD4+CD8+ T cells (CD3+CD21-IgM-CD4+CD8+), CD8+ T cells (CD3+CD21-IgM-CD4-CD8+), and B cells (CD3-CD21+IgM+CD4-CD8-) where analyzed in parallel from those two independent FSClow and FSChigh gates. The representative examples on the right side clearly show that proliferative cells are CellTracelow FSChigh. (C, D) At day 56 post-first immunization, freshly isolated PBMCs and LN cells where exposed against recombinant CSFV E2 and influenza virus HA and NP, or were let unstimulated (“-”). For all samples, Con A was used as a positive control. (C) Induction of FSChigh cell population following Ag restimulation. Cells from the individual animals are represented by separate symbols. The percentage of FSChigh cells was determined by flow cytometry with FlowJo. (*p ≤…

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  Figure 4. Virus replicon particle (VRP) and polyplex induce a strong and specific CD4+ T cell immune response against influenza antigens. Induction of CD4+ T cell immune responses against the RepRNA encoded CSFV E2 and influenza virus HA and NP, determined by restimulation of freshly isolated PBMCs and LN cells (day 56 post-first immunization); cells were restimulated with recombinant E2 (1 µg/ml), HA (1 µg/ml) or NP (1 µg/ml), or non-stimulated (-). (A) After 5 days of cell culture, the numbers of division cycles (D0-D7) of proliferating CD4+ T cells in PBMC and draining LN compartments, were determined by flow cytometry using the FlowJo software. The values represent the mean of proliferating CD4+ T cells of the five individual pigs for each group. Numbers in red represent the mean percentage of highly proliferative cells (≥ 5 division cycles). (B) After 5 days of cell culture, representative dot plots are shown for draining LN compartments of three immunized animals per groups for proliferating CD4+ T cells.

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  Figure 5. Virus replicon particle (VRP) and polyplex induce a specific CD8+ T cell immune response against influenza antigens. Induction of CD8+ T cell immune responses against the RepRNA encoded CSFV E2 and influenza virus HA and NP, determined by restimulation of freshly isolated PBMCs and cells derived from draining LNs (day 56 post-first immunization); cells were restimulated with recombinant E2 (1 µg/ml), HA (1 µg/ml) or NP (1 µg/ml), or non-stimulated (-). (A) After 5 days of cell culture, the numbers of division cycles (D0-D7) of proliferating CD8+ T cells in PBMC and draining LN compartments, were determined by flow cytometry using the FlowJo software. The values represent the mean of proliferating CD8+ T cells of the five individual pigs for each group. Numbers in red represent the mean percentage of highly proliferative cells (≥ 5 division cycles). (B) After 5 days of cell culture, representative dot plots are shown for draining LN compartments of three immunized animals per group for proliferating CD8+ T cells.

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  Figure 6. Virus replicon particle (VRP) and polyplex activate CD4+CD8+ T cells response against influenza antigens. Induction of CD4+CD8+ T cell immune responses against the RepRNA encoded CSFV E2 and influenza virus HA and NP, determined by restimulation of freshly isolated PBMCs and cells derived from draining LNs (day 56 post-first immunization); cells were restimulated with recombinant E2 (1 µg/ml), HA (1 µg/ml) or NP (1 µg/ml), or non-stimulated (-). (A) After 5 days of cell culture, the numbers of division cycles (D0-D7) of proliferating CD4+ T cells in PBMC and draining LN compartments, were determined by flow cytometry using the FlowJo software. The values represent the mean of proliferating CD4+CD8+ T cells of the five individual pigs for each group. Numbers in red represent the mean percentage of highly proliferative cells (≥ 5 division cycles). (B) After 5 days of cell culture, representative dot plots are shown for draining LN compartments of three immunized animals per group for proliferating CD4+CD8+ T cells. (C) For the counts of CD4+CD8+ T cell subset, we calculated the percentage of total events: ratio (number of events in the gated cell subtype:…

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  Figure 7. Virus replicon particle (VRP) induce a strong and specific humoral immune response against influenza antigens. Induction of B cell immune responses against the RepRNA encoded CSFV E2 and influenza virus HA and NP, determined by restimulation of freshly isolated PBMCs and cells derived from draining LNs (day 56 post-first immunization); cells were restimulated with recombinant E2 (1 µg/ml), HA (1 µg/ml) or NP (1 µg/ml), or non-stimulated (-). (A) After 5 days of cell culture, the numbers of division cycles (D0-D7) of proliferating B cells in PBMC and draining LN compartments, were determined by flow cytometry using the FlowJo software. The values represent the mean of proliferating B cells of the five individual pigs for each group. Numbers in red represent the mean percentage of highly proliferative cells (≥ 5 division cycles). (B) After 5 days of cell culture, representative dot plots are shown for immunized animals per group for proliferating B cells. (C) Induction of humoral immune response (IgG titer) against RepRNA encoded influenza virus HA and NP, determined by ELISA using day -5 (baseline) and day 56 (post-boost 2) sera from individual pigs. ***p < 0.001; *p < 0.05.