The HSP70-fused foot-and-mouth disease epitope elicits cellular and humoral immunity and drives broad-spectrum protective efficacy

Abstract

Current foot-and-mouth disease (FMD) vaccines have significant limitations, including side effects due to oil emulsions at the vaccination site, a narrow spectrum of protective efficacy, and incomplete host defenses mediated by humoral immunity alone. To overcome these limitations, new FMD vaccines must ensure improved safety with non-oil-based adjuvants, a broad spectrum of host defenses within/between serotypes, and the simultaneous induction of cellular and humoral immunity. We designed a novel, immune-potent, recombinant protein rpHSP70-AD that induces robust cellular immunity and elicits a broad spectrum of host defenses against FMD virus (FMDV) infections. We demonstrated that an oil emulsion-free vaccine containing rpHSP70-AD mediates early, mid-term, and long-term immunity and drives potent host protection against FMDV type O and A, suggesting its potential as an FMD vaccine adjuvant in mice and pigs. These results suggest a key strategy for establishing next-generation FMD vaccines, including novel adjuvants.

Fig. 1. A schematic diagram of the novel immunopotent porcine recombinant protein, rpHSP70-AD, and expression of rpHSP70-AD.

Schematic diagram of the novel immunopotent porcine recombinant protein, rpHSP70-AD (a); The novel immunopotent recombinant protein rpHSP70-AD combines the multiple indicated active immunopotent domains. The detailed strategy is described in Methods, and the sequence information for each molecule is described in Supplementary Table 1. Sun-Gel-stained SDS-PAGE gel of rpHSP70-AD, M; molecular size marker, E; purified elution fraction (b); Western blot of purified recombinant rpHSP70-AD, T; total cell lysates, S; soluble fraction of cell lysates, E; purified elution fraction (c).

Fig. 2. rpHSP70-AD mediated cytokine expression and temporal kinetics in vivo.

C57BL/6 mice (n = 5/group/time point) were injected intraperitoneally with 50 μg rpHSP70-AD and sacrificed at 0, 6, 12, and 24 h post-injection. Peritoneal lavage fluid was collected by washing the peritoneal cavity with 2 mL chilled DPBS. Samples were then centrifuged, and the supernatant was analyzed for cytokines using ELISA. Cytokine profiles and temporal kinetics in vivo induced by rpHSP70-AD: IL-1β (a); IL-2 (b); IL-4 (c); IL-6 (d); IL-9 (e); IL-10 (f); IL-12/23p40 (g); IL-15/15 R (h); IL-17A (i); IL-21 (j); IL-22 (k); IFNγ (l). The data are represented as the mean ± SEM of triplicate measurements (n = 5). Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test. ^*p < 0.05, ^***p < 0.001.

Fig. 3. rpHSP70-AD induced a broad spectrum of host protection against FMDV O (O/VET/2013) and FMDV A (A/Malay/97) infection in mice.

C57BL/6 mice (n = 5/group) were administered with test vaccine including 10 μg rpHSP70-AD, ISA 206 (oil-based emulsion, 50%, w/w), 10% Al(OH)₃, and 15 μg Quil-A. A negative control (NC) group was injected with the same volume of PBS. The test vaccines were injected intramuscularly into mice that were later challenged with FMDV O (100 LD₅₀ O/VET/2013) and FMDV A (100 LD₅₀ A/Malay/97) at 7 days post-vaccination. The survival rates and body weights were monitored for 7 days post-challenge. Experimental strategy (a); survival rates post-challenge with O/VET/2013 and A/Malay/97 (b); changes in body weight post-challenge with O/VET/2013 and A/Malay/97 (c). The data represent the mean ± SEM of triplicate measurements (n = 5/group). Statistical analyses were performed using two-way ANOVA with Bonferroni correction. ^*p < 0.05; ^**p < 0.01.

Fig. 4. rpHSP70-AD mediated early, mid-term, and long-term immune responses and host defense against FMDV infection in mice.

C57BL/6 mice (n = 5/group) were administered oil emulsion-free test vaccines, including O TWN antigen, A22 antigen, or combined O TWN and A22 antigens with rpHSP70-AD. The positive control received 1.5 μg (1/10 dose for cattle and pig use) O TWN antigen or A22 antigen without (w/o) oil emulsion, 10% Al(OH)₃, and 15 μg Quil-A. The negative control received the same volume of PBS. Vaccination was performed twice at 35-day intervals. Study strategy (a); antibody titers by SP O ELISA (b); O/TWN/97 VN titers (c); antibody titers by SP A ELISA (d); A22/IRAQ VN titers (e); survival rate at 84 days post-vaccination challenge with FMDV O (O/VET/2013) (f); changes in body weight at 84 days post-vaccination challenge with FMDV O (O/VET/2013) (g); survival rate at 84 days post-vaccination challenge with FMDV A (A/Malay/97) (h); changes in body weight at 84 days post-vaccination challenge with FMDV A (A/Malay/97) (i); survival rate at 168 days post-vaccination challenge with FMDV O (O/VET/2013) (j); changes in body weight at 168 days post-vaccination challenge with FMDV O (O/VET/2013) (k); survival rate at 168 days post-vaccination challenge with FMDV A (A/Malay/97) (l); changes in body weight at 168 days post-vaccination challenge with FMDV A (A/Malay/97) (m). The data represent the mean ± SEM of triplicate measurements (n = 5/group). Statistical analyses involved two-way ANOVA with Bonferroni correction. ⁺p < 0.05; ⁺⁺p < 0.01; and ⁺⁺⁺p < 0.001: NC vs PC (O TWN- or A22-only group); ^#p < 0.05; ^##p < 0.01; and ^###p < 0.001: NC vs Exp. (O TWN + rpHSP70-AD or A22 + rpHSP70-AD group); ^*p < 0.05; ^**p < 0.01; and ^***p < 0.001: PC vs Exp.

Fig. 5. rpHSP70-AD induced porcine PBMC cell proliferation and cytokine expression.

Porcine PBMCs were co-incubated with rpHSP70-AD (final concentration: 2 μg/mL). Exactly 0 and 24 h after co-incubation, cell proliferation was tested using the BrdU ELISA kit. Cell culture supernatants were harvested for cytokine ELISA. In vitro cell proliferation induced by rpHSP70-AD in porcine PBMCs (a); IL-1β (b); IL-2 (c); IL-6 (d); IL-10 (e); IL-12/23p40 (f); IL-17A (g); and IFNγ (h) expression induced by rpHSP70-AD in porcine PBMCs. The data are represented as the mean ± SEM of triplicate measurements (n = 3). Statistical analyses were performed using unpaired two-tailed Student’s t-test. ^***p < 0.001.

Fig. 6. rpHSP70-AD mediates early, mid-term, and long-term immune responses in pigs.

For pig experiments, FMD antibody-seronegative animals (10–12 weeks old) were used. Pigs were divided into two groups (n = 5/group) and administrated oil emulsion-free test vaccines, including A22 antigen alone, or combined A22 antigens with rpHSP70-AD. The positive control group received 15 μg (one dose for cattle and pig use) A22 antigen without (w/o) oil emulsion, 10% Al(OH)₃, and 150 μg Quil-A. The vaccination was performed twice at 28-day intervals, with 1 mL vaccine (one dose) injected via a deep intramuscular route on the animals’ necks. Blood samples were collected at 0, 7, 14, 28, 42, 56, 70, and 84 days post-vaccination in pigs for serological assays. Study strategy (a); SP A antibody titers (b); and A22/IRAQ VN titers (c). The data represent the mean ± SEM of triplicate measurements (n = 5/group). Statistical analyses were performed using two-way ANOVA with Bonferroni correction. ^*p < 0.05; ^**p < 0.01; and ^***p < 0.001.

Fig. 7. rpHSP70-AD mediates host defense against FMDV O and A infection in pigs.

For the challenge experiments, pigs (10–12 weeks old, n = 3–5/group) were administered oil emulsion-free test vaccines, including O BE + A YC antigen alone or combined O BE + A YC antigens with rpHSP70-AD. Vaccinated pigs were challenged with FMDV type O (O/SKR/JC/2014) or FMDV type A (A/SKR/GP/2018) on the heel bulb at 10⁵ TCID₅₀/100 μL. Study strategy (a); SP O and SP A antibody titers (b); O/SKR/BE/2017 and A/SKR/YC/2017 VN titers (c); clinical score and the amount of virus in serum and oral swab from negative control (n = 3/group) pigs against FMDV type O (O/SKR/JC/2014) (d) or FMDV type A (A/SKR/GP/2018) (g); clinical score and the amount of virus in serum and oral swab from positive control (O BE + A YC, n = 3/group) pigs against FMDV type O (O/SKR/JC/2014) (e) or FMDV type A (A/SKR/GP/2018) (h); clinical score and the amount of virus in serum and oral swab from experimental group (O BE + A YC + rpHSP70-AD, n = 5/group) pigs against FMDV type O (O/SKR/JC/2014) (f) or FMDV type A (A/SKR/GP/2018) (i). The left Y-axis of the graph shows the amount of virus in sera and oral swab as log₁₀ values; the right Y-axis shows the clinical index as the maximum value of 10 points. The data represent the mean ± SEM of triplicate measurements (n = 3∼5/group). Statistical analyses involved two-way ANOVA with Bonferroni correction. ^***p < 0.001: NC vs PC, NC vs Exp.; ^#p < 0.05; ^##p < 0.01; ^###p < 0.001: PC vs Exp.