Prophylactic treatment with PEGylated bovine IFNλ3 effectively bridges the gap in vaccine-induced immunity against FMD in cattle

Abstract

Foot-and-mouth disease (FMD) is a vesicular disease of cloven-hoofed animals with devastating economic implications. The current FMD vaccine, routinely used in enzootic countries, requires at least 7 days to induce protection. However, FMD vaccination is typically not recommended for use in non-enzootic areas, underscoring the need to develop new fast-acting therapies for FMD control during outbreaks. Interferons (IFNs) are among the immune system's first line of defense against viral infections. Bovine type III IFN delivered by a replication defective adenovirus (Ad) vector has effectively blocked FMD in cattle. However, the limited duration of protection-usually only 1-3 days post-treatment (dpt)-diminishes its utility as a field therapeutic. Here, we test whether polyethylene glycosylation (PEGylation) of recombinant bovine IFNλ3 (PEGboIFNλ3) can extend the duration of IFN-induced prevention of FMDV infection in both vaccinated and unvaccinated cattle. We treated groups of heifers with PEGboIFNλ3 alone or in combination with an adenovirus-based FMD O1Manisa vaccine (Adt-O1M) at either 3 or 5 days prior to challenge with homologous wild type FMDV. We found that pre-treatment with PEGboIFNλ3 was highly effective at preventing clinical FMD when administered at either time point, with or without co-administration of Adt-O1M vaccine. PEGboIFNλ3 protein was detectable systemically for >10 days and antiviral activity for 4 days following administration. Furthermore, in combination with Adt-O1M vaccine, we observed a strong induction of FMDV-specific IFNγ+ T cell response, demonstrating its adjuvanticity when co-administered with a vaccine. Our results demonstrate the promise of this modified IFN as a pre-exposure prophylactic therapy for use in emergency outbreak scenarios.

Keywords: FMDV; IFN; IFNλ3; IL28B; PEGylation; biotherapeutics; foot-and-mouth disease; type III interferon.

Figure 1

(A) Virus yield reduction assay of recombinant boIFNλ3 or PEGboIFNλ3 against VSV NJ. MDBK-t2 cells were treated with 2-fold dilutions of boIFNλ3 or PEGboIFNλ3. After overnight incubation, cells were challenged with VSV NJ at MOI of 0.1 and incubated for 48 h. Titers of VSV were evaluated by TCID₅₀ and expressed as relative titer as compared to untreated cells. Average data points from duplicate measurements are represented. A sigmoidal dose–response curve was fitted to determine IC₅₀ values for each recombinant IFN. (B) In vitro antiviral activity of recombinant boIFNλ3 vs. recombinant PEGboIFNλ3 against FMDV SAT1. MDBK cells were treated with 2-fold dilutions of boIFNλ3 or PEGboIFNλ3. After overnight incubation, cells were challenged with FMDV SAT1 at MOI of 0.1 and incubated for another 48 h_. Titers of FMDV were evaluated in the cell supernatants by end point dilution on BHK-21 cells.

Figure 2

(A) Four-to-six-month-old Holstein-Fresian calves were injected with 75 or 150 μg/kg PEGboIFNλ3 and blood was collected at various time points for pharmacokinetic analysis. (B) PEGboIFNλ3 concentration in the serum was measured on the Mesoscale Discovery (MSD) platform via an electrochemiluminescent assay (ECLA). (C) Serum antiviral activity was measured via Mx CAT ELISA on cattle serum from the pharmacokinetic study. n = 2 cattle/sex/dose.

Figure 3

(A) Holstein calves of approximately 450 lb were subcutaneously injected with 150 μg/kg PEGboIFNλ3 and/or 2.5 × 10⁹ pfu Adt-O1M FMD vaccine at either 3 or 5 days prior to intranasopharyngeal challenge with 2 × 10⁶ BID₅₀ FMDV O1Manisa. A control group was inoculated at 3 days prior to challenge with 2.5 × 10⁹ pfu Ad5-Blue. Blood was collected daily after treatment and serum and purified peripheral blood mononuclear cells (PBMCs) were preserved for later testing. (B) Serum antiviral activity was assessed by Mx CAT ELISA. (C) Interferon stimulated gene (ISG) induction was assessed in purified PBMCs daily following treatment. Change in gene expression is represented as the mean fold induction of each gene compared to the baseline pre-treatment time point, shaded according to intensity of up-or down-regulation of the gene. n = 2–3 calves/treatment group/time point.

Figure 4

Holstein calves of approximately 450 lb were subcutaneously injected with 150 μg/kg PEGboIFNλ3 and/or 2.5 × 10⁹ pfu Adt-O1M FMD vaccine at either 3 or 5 days prior to intranasopharyngeal challenge with 2 × 10⁶ BID₅₀ FMDV O1Manisa. A control group was inoculated 3 days prior to challenge with 2.5 × 10⁹ pfu Ad5-Blue. Cattle were assessed for clinical score (bars) on days 3, 4, 6, and 8 post-challenge and EDTA-treated blood was assessed for signs of lymphopenia daily (dotted line). n = 2–3 cattle/time point/treatment group.

Figure 5

Holstein calves of approximately 450 lb were subcutaneously injected with 150 μg/kg PEGboIFNλ3 and/or 2.5 × 10⁹ pfu Adt-O1M FMD vaccine at either 3 or 5 days prior to intranasopharyngeal challenge with 2 × 10⁶ BID₅₀ FMDV O1Manisa. A control group was inoculated 3 days prior to challenge with 2.5 × 10⁹ pfu Ad5-Blue. Daily, from 0 till 8 days post-challenge, serum and nasal swabs were collected and assessed for presence of FMDV. Viremia is reported in both PFU/mL of serum (solid red line) and GCN/mL of serum (dotted red line). Virus shedding is expressed in both PFU/mL of nasal secretions (solid blue line) and GCN/mL in nasal secretions (dotted blue line). n = 2–3 calves/treatment group/time point.

Figure 6

Holstein calves of approximately 450 lb were subcutaneously injected with 150 μg/kg PEGboIFNλ3 and/or 2.5 × 10⁹ pfu Adt-O1M FMD vaccine at either 3 or 5 days prior to intranasopharyngeal challenge with 2 × 10⁶ BID₅₀ FMDV O1Manisa. A control group was inoculated 3 days prior to challenge with 2.5 × 10⁹ pfu Ad5-Blue. Blood was collected daily after treatment and challenge and serum and peripheral blood mononuclear cells (PBMCs) purified and preserved for later testing. n = 2–3 calves/treatment group/time point. (A) Heat-inactivated serum was tested at various time points for FMDV O1Manisa virus neutralizing titer. Titers expressed as the Log₁₀ TCID₅₀/mL of serum. ^*p value < 0.05 compared to the control group at the given time-point. (B) Isolated PBMCs were stained for flow cytometric analysis. Upon ex vivo stimulation with MOI 2 FMDV O1Manisa, the induction of IFNγ expression in CD4-CD8+ and CD4 + CD8-T cell populations was measured and expressed as the difference in percent of the single positive T cell parent population between stimulated and unstimulated wells. (C) Adaptive immunity-related gene induction was assessed in PBMCs at various time points following challenge. Change in gene expression is represented as the mean fold induction of each gene compared to the baseline pre-treatment time point, shaded according to intensity of up-or down-regulation of the particular gene. ^*p value < 0.05 compared to within-group 0 dpi ^**p value < 0.01 compared to within-group 0 dpi ^***p value < 0.001 compared to within-group 0 dpi.