Immunogenicity of chimeric hemagglutinins delivered by an orf virus vector platform against swine influenza virus

Abstract

Orf virus (ORFV) is a large DNA virus that can harbor and efficiently deliver viral antigens in swine. Here we used ORFV as a vector platform to deliver chimeric hemagglutinins (HA) of Influenza A virus of swine (IAV-S). Vaccine development against IAV-S faces limitations posed by strain-specific immunity and the antigenic diversity of the IAV-S strains circulating in the field. A promising alternative aiming at re-directing immune responses on conserved epitopes of the stalk segment of the hemagglutinin (HA2) has recently emerged. Sequential immunization with chimeric HAs comprising the same stalk but distinct exotic head domains can potentially induce cross-reactive immune responses against conserved epitopes of the HA2 while breaking the immunodominance of the head domain (HA1). Here, we generated two recombinant ORFVs expressing chimeric HAs encoding the stalk region of a contemporary H1N1 IAV-S strain and exotic heads derived from either H6 or H8 subtypes, ORFV^Δ121cH6/1 and ORFV^Δ121cH8/1, respectively. The resulting recombinant viruses were able to express the heterologous protein in vitro. Further, the immunogenicity and cross-protection of these vaccine candidates were assessed in swine after sequential intramuscular immunization with OV-cH6/1 and OV-cH8/1, and subsequent challenge with divergent IAV-S strains. Humoral responses showed that vaccinated piglets presented increasing IgG responses in sera. Additionally, cross-reactive IgG and IgA antibody responses elicited by immunization were detected in sera and bronchoalveolar lavage (BAL), respectively, by ELISA against different viral clades and a diverse range of contemporary H1N1 IAV-S strains, indicating induction of humoral and mucosal immunity in vaccinated animals. Importantly, viral shedding was reduced in nasal swabs from vaccinated piglets after intranasal challenge with either Oh07 (gamma clade) or Ca09 (npdm clade) IAV-S strains. These results demonstrated the efficiency of ORFV-based vectors in delivering chimeric IAV-S HA-based vaccine candidates and underline the potential use of chimeric-HAs for prevention and control of influenza in swine.

Keywords: ORFV vectors; chimeric HA; cross-protection; swine influenza; vaccines.

Figure 1

Overview of the chimeric vaccine strategy. (A) Sequential immunization with the chimeric HA constructs composed of exotic heads and sharing the same stalk domain belonging to the H1 subtype. The rationale for this approach is breaking the immunodominance of HA head by inducing a recall response against the conserved stalk, which may generate cross-reactive antibodies capable of protecting swine against divergent IAV-S. (B) HA-based phylogenetic tree representing the subclassification of influenza A virus subtypes into two groups (group 1 and 2). The arrows indicate the subtypes from group 1 used to design the head of the chimeric HAs containing a contemporary H1 stalk domain. (C) Representation of the construction of the recombinant plasmid for the generation of ORFV^Δ121cH6/1 recombinant virus through homologous recombination. (D) Representation of the construction of the recombinant plasmid for the generation of ORFV^Δ121cH8/1 recombinant virus through homologous recombination.

Figure 2

Characterization of ORFV^Δ121cH6/1 and ORFV^Δ121cH8/1. (A) Agarose gel demonstrating that an H1 stalk fragment was amplified, confirming its presence in both recombinant viruses, and the lack of amplification of ORF121 gene, indicating that the recombinant ORFV^Δ121cH6/1 and ORFV^Δ121cH8/1 viruses were purified. Growth curves comparing the replication of recombinant (B) ORFV^Δ121cH6/1 and (C) ORFV^Δ121cH8/1 viruses and the parental OV-IA82 virus at different time points in OFTu (left) and in STu (right) cells.

Figure 3

Expression of heterologous proteins by ORFV^Δ121cH6/1 and ORFV^Δ121cH8/1 recombinant viruses. Expression of chimeric HAs (red fluorescence) by the recombinant (A) ORFV^Δ121cH6/1 and (B) ORFV^Δ121cH8/1 viruses was assessed by IFA in permeabilized (left) and non-permeabilized cells (right) infected with 1 MOI (48 hpi). (C) Increasing levels of cH6/1 (~70 kDa) and (D) cH8/1 in ORFV^Δ121cH6/1-infected and ORFV^Δ121cH8/1-infected ovine cells, respectively, as detected by Western blot. Mock-infected OFTu cells were used as negative controls, while beta actin was used as loading control for the WB. (E) The flow cytometry data were consistent with the two previous assays, showing a slightly higher expression of cH6/1 than (F) cH8/1 at 72 hpi. OV-IA82-infected OFTu cells were used as negative controls for the gating strategy, where the gated cells indicate positive cell population.

Figure 4

Dynamics of antibody responses to immunization. Increasing IgG responses induced by priming with ORFV^Δ121cH6/1 and booster with ORFV^Δ121cH8/1-IAV-S in Oh07 and Ca09 challenged pigs were assessed by whole-virus ELISA for the challenge virus (A) Oh07 and (B) Ca09 at the indicated time points post-immunization. p-values: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 5

Humoral responses to immunization at day 35 was assessed by whole-virus ELISA against a panel of 10 divergent viruses. IgG antibody levels by prime with ORFV^Δ121cH6/1 and booster with ORFV^Δ121cH8/1-IAV-S in (A) Oh07 and (B) Ca09 challenged animals. p-values: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 6

Mucosal antibody responses to immunization were assessed in BAL by whole-virus ELISA against a panel of 10 divergent viruses at D42. IgA antibody levels induced by prime with ORFV^Δ121cH6/1 and booster with ORFV^Δ121cH8/1 in (A) Oh07 and (B) Ca09 challenged animals is shown in blue. p-values: *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 7

Shedding of IAV-S through nasal secretions. Viral shedding in (A) Oh07 challenged pigs and (B) Ca09 challenged pigs was determined by infectious titer (upper) and (lower) genome copy numbers. p-values: *p < 0.05 and **p < 0.01.

Figure 8

Lung scores and viral load in target tissues. (A) Lung lesions and presence of genomic RNA locally in swine challenged with Oh07. (B) Similarly, lung lesion scores and viral load were evaluated in Ca09 challenged animals. p-values: *p < 0.05 and **p < 0.01.