Protective Efficacy of an Orf Virus-Vector Encoding the Hemagglutinin and the Nucleoprotein of Influenza A Virus in Swine

Abstract

Swine influenza is a highly contagious respiratory disease of pigs caused by influenza A viruses (IAV-S). IAV-S causes significant economic losses to the swine industry and poses challenges to public health given its zoonotic potential. Thus effective IAV-S vaccines are needed and highly desirable and would benefit both animal and human health. Here, we developed two recombinant orf viruses, expressing the hemagglutinin (HA) gene (OV-HA) or the HA and the nucleoprotein (NP) genes of IAV-S (OV-HA-NP). The immunogenicity and protective efficacy of these two recombinant viruses were evaluated in pigs. Both OV-HA and OV-HA-NP recombinants elicited robust virus neutralizing antibody response in pigs, with higher levels of neutralizing antibodies (NA) being detected in OV-HA-NP-immunized animals pre-challenge infection. Although both recombinant viruses elicited IAV-S-specific T-cell responses, the frequency of IAV-S-specific proliferating CD8+ T cells upon re-stimulation was higher in OV-HA-NP-immunized animals than in the OV-HA group. Importantly, IgG1/IgG2 isotype ELISAs revealed that immunization with OV-HA induced Th2-biased immune responses, whereas immunization with OV-HA-NP virus resulted in a Th1-biased immune response. While pigs immunized with either OV-HA or OV-HA-NP were protected when compared to non-immunized controls, immunization with OV-HA-NP resulted in incremental protection against challenge infection as evidenced by a reduced secondary antibody response (NA and HI antibodies) following IAV-S challenge and reduced virus shedding in nasal secretions (lower viral RNA loads and frequency of animals shedding viral RNA and infectious virus), when compared to animals in the OV-HA group. Interestingly, broader cross neutralization activity was also observed in serum of OV-HA-NP-immunized animals against a panel of contemporary IAV-S isolates representing the major genetic clades circulating in swine. This study demonstrates the potential of ORFV-based vector for control of swine influenza virus in swine.

Keywords: cell-mediated immunity; neutralizing antibodies; orf virus; swine influenza virus; vectored-vaccine.

Figure 1

Construction of ORFV recombinants and their replication kinetics. (A) Schematic representation of homologous recombination between pUC57-121LR-SIV-HA-loxp-GFP plasmid and ORFV-IA82 genome. The recombinant virus was treated with Cre recombinase to remove GFP marker gene to obtain markerless OV-HA construct. (B) Schematic representation of homologous recombination between pUC57-127LR-SIV-NP-loxp-GFP plasmid and OV-HA genome. The recombinant virus was treated with Cre recombinase to obtain markerless OV-HA-NP construct. (C) Multi-step (0.1 MOI) and single step (10 MOI) growth curve of OV-HA and OV-HA-NP. OFTu or STU cells were infected with OV-HA and-HA-NP recombinants and virus titers were calculated at 0, 6, 12, 24, 48 and 72 hours post-infection. Error bars represent SEM calculated based on three independent experiments.

Figure 2

Expression of heterologous proteins by ORFV recombinants. (A) Immunofluorescence assay in permeabilized OFTu cells. Upper panel shows expression of HA protein and absence of NP protein in OV-HA recombinant. Lower panel shows expression of HA and NP protein by OV-HA-NP recombinant. (B) Immunofluorescence assay performed in non-permeabilized OFTu cells. Upper panel shows expression of HA by OV-HA recombinant and lower panel shows expression of HA and NP by OV-HA-NP recombinant. Blue fluorescence in merged images in panel A and B indicates nuclear staining by DAPI. (C) Expression of heterologous proteins by ORFV recombinants assessed by flow-cytometry. OFTu cells were infected with OV-HA, OV-HA-NP or Wild-type OV-IA82 as negative control. Infected cells were collected 48 hours post-infection, fixed and then stained with appropriate antibodies for flow cytometric analysis.

Figure 3

Immunization-challenge experiment design and humoral response to immunization. (A) A timeline of immunization-challenge experiment. (B) IAV-S specific neutralizing antibody response elicited by immunization with OV-HA and OV-HA-NP. (C) VN titer of individual animal measured on 0 dpc (pre-challenge) and 7 dpc (post-challenge). (D) IAV-S specific humoral immune response induced by OV-HA and OV-HA-NP assessed by hemagglutination inhibition (HI) assay. Red arrow heads represent the day of challenge. The error bars represent SEM. VN titer shown in logarithmic scale for effective visualization. HI titer shown in liner scale. P-values: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) HI titer of individual animal measured on 0 dpc (pre-challenge) and 7 dpc (post-challenge). Wilcoxson rank sum test was used for comparing means (pre-challenge vs. post-challenge) within the groups. *P < 0.05; **P < 0.01; ns, non-significant.

Figure 4

IAV-S specific IgG responses to immunization. (A) Total serum IgG level elicited by OV-HA and OV-HA-NP immunization at various time points were assessed by ELISA. Isotype ELISA demonstrating endpoint titers elicited by immunization at 35 days pi in serum was assessed for detecting specific (B) IgG1 and (C) IgG2 antibodies. (D) IgG1/IgG2 ratio in immunized animals. Each dot represents IgG1/IgG2 ratio of an individual animal. Middle bar represents mean ratio and upper and lower bars represent range. P-values: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, non-significant.

Figure 5

T-cell immune response to immunization. PBMCs isolated from pigs at 35 dpi following recall stimulation with inactivated IAV-S were analyzed for: (A) IFN-γ production by different T-cell subsets measured by flow cytometry assay; and (B) T-cells proliferation by CFSE dilution assay. Data represents group means and error bars represent SEM. P-values: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, non-significant.

Figure 6

Protective efficacy of OV-HA and OV-HA-NP against IAV-S challenge. (A) IAV-S viral RNA shedding in the nasal swab was determined by RT-qPCR and expressed as log10 genome copy number per milliliter. (B) IAV-S viral load in the lung determined by RT-qPCR and expressed as log10 genome copy number per milliliter. Data represents group mean and error bars represent SEM. P-values: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, non-significant.

Figure 7

Cross-neutralization assay. (A) Phylogenetic tree based on HA amino acid sequence. Animo acid sequences were aligned using MUSCLE and trees were constructed using Maximum Likelihood method implemented in MEGA-X (53). (B) Phylogenetic tree based on amino acid sequence of NP protein. Sequences were aligned using MUSCLE and trees were constructed using Maximum Likelihood method. (C) Cross-neutralizing antibody titers against different IAV-S viruses in the serum samples collected on 35 days post-immunization. Each panel represents one virus isolate and the percentage of its HA/NP similarity with that of vaccine HA/NP is provided under the name of each isolate. Bar represent mean and error bar represent SEM. P-values: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.