Chimeric calicivirus-like particles elicit specific immune responses in pigs

Abstract

Virus-like particles (VLPs) have received considerable attention due to their potential application in veterinary vaccines and, in particular, VLPs from rabbit haemorrhagic disease virus (RHDV) have successfully shown to be good platforms for inducing immune responses against an inserted foreign epitope in mice. The aim of this study was to assess the immunogenicity of chimeric RHDV-VLPs as vaccine vectors in pigs. For this purpose, we have generated chimeric VLPs containing a well-known T epitope of 3A protein of foot-and-mouth disease virus (FMDV). Firstly, RHDV-VLPs were able to activate immature porcine bone marrow-derived dendritic cells (poBMDCs) in vitro. Secondly, pigs were inoculated twice in a two-week interval with chimeric RHDV-VLPs at different doses intranasally or intramuscularly. One intramuscularly treated group was also inoculated with adjuvant Montanide™ ISA 206 at the same time. Specific IgG and IgA antibodies against RHDV-VLPs were induced and such levels were higher in the adjuvanted group compared with other groups. Interestingly, anti-RHDV-VLP IgA responses were higher in groups inoculated intramuscularly than those that received the VLPs intranasally. Two weeks after the last immunisation, specific IFN-γ-secreting cells against 3A epitope and against RHDV-VLPs were detected in PBMCs by ELISPOT. The adjuvanted group exhibited the highest IFN-γ-secreting cell numbers and lymphoproliferative specific T cell responses against 3A epitope and RHDV-VLP. This is the first immunological report on the potential use of chimeric RHDV-VLPs as antigen carriers in pigs.

Fig. 1

Phenotypic maturation of poBMDCs after exposure to RHDV-VLPs or LPS. PoBMDCs were incubated with either RHDV-VLPs (50 μg/ml) or LPS (10 μg/ml) and after 24 h of culture, surface expression of SLA-II, CD80/86 and CD86 was determined by flow cytometry using PE-conjugated secondary mAb. (A) Dot-plot shows the gating strategy based on FSC/SSC. R0 region indicates poBMDCs. (B) Histograms show expression patterns of poBMDCs gated for FS/SS. RHDV-VLP-pulsed poBMDCs (solid line) and LPS-exposed poBMDCs (dotted line) express higher levels of all markers as compared to un-stimulated poBMDCs in steady state (grey histograms) or isotype control (black histograms). Results of one representative experiment of a minimum of five independent experiments are shown.

Fig. 2

Cytokine production of poBMDCs pulsed with VLPs and LPS or poly:IC in vitro. Immature poBMDCs were pulsed with RHDV-VLPs (50 μg/ml) or LPS (10 μg/ml) or poly:IC (50 μg/ml). After 4, 8, 16 and 24 h of culture, secreted TNF-α and IL-6 were quantified by commercially available ELISAs. Cytokine levels in culture supernatants are shown in pg/ml (mean ± standard deviation). As controls, poBMDCs were cultured in medium alone (Mock). The limit of detection was around 148 pg/ml for TNF-α and 70 pg/ml for IL-6. Results of triplicate wells of one representative experiment of a minimum of three independent experiments are shown.

Fig. 3

Specific humoral responses of RHDV-3A-VLP immunised pigs against the vector RHDV-VLP in serum and saliva at day 14 after the second immunisation. (A) Anti-RHDV-VLP total Ig antibodies in serum. (B) Anti-RHDV-VLP IgG1 antibodies in serum. (C) Anti-RHDV-VLP IgG2 antibodies in serum. (D) Anti-RHDV-VLP IgA antibodies in serum. (E) Anti-RHDV-VLP IgA antibodies in saliva. Pigs are divided in different groups depending on the inoculation route: B (IN) (grey line), C (IM + ADJ) (solid black line) and D (IM) (dotted line). Pigs are also divided depending on the VLP dose: 20 μg (triangle), 60 μg (rhomb) and 180 μg (square), summarised in the legend. Titres are expressed as reciprocals of the last dilution of sera (log₁₀), calculated by interpolation to give an A₄₉₂ of 1.0 OD unit. Each value corresponds to geometric mean of all the animals (duplicate wells) of each group.

Fig. 4

Specific cellular responses of RHDV-3A-VLP immunised pig against the vector RHDV-VLP, against the peptide 3A and against chimeric RHDV-3A-VLP at day 28. Pigs are divided in different groups, depending on the inoculation route: intranasal (IN), intramuscular + adjuvant (IM + ADJ) and intramuscular (IM). Specific RHDV-VLP and 3AT IFN-γ-producing cells are detected by ELISPOT (A). The background values (number of spots in negative control wells) were subtracted from the respective counts of the stimulated cells and the immune responses were expressed as number of spots per million of PBMCs for each animal. Shown are the results of duplicate wells of one representative experiment. Specific RHDV-VLP (B), 3A (B) and RHDV-3A-VLP (C) T-cell proliferation is detected by lymphoproliferation assay. Data are shown as SI (stimulation indexes, see Section 2) of each animal. Results of triplicate wells of one representative experiment are shown.

Fig. 5

Histopathological analysis of injection point (brachiocephalicus muscle) of pigs at day 14 after the second immunisation (H–E stain, bar = 200 μm). Lesions were classified as (A) absence; (B) mild: small focus of granulomatous inflammation located in the perimuscular adipose tissue; (C) moderate: granulomatous reaction is partially occupying the perimuscular fat with mild infiltration of the adjacent muscular tissue; (D) severe: adipose and muscular tissues are infiltrated and almost substituted by a diffuse granulomatous inflammation. (E) In the graphic is shown the pathological score (0–3) for all the groups indicated as mean (bars) ± standard deviation (lines).

Fig. 6

Phenotypic maturation of huMoDCs after exposure to RHDV-VLPs or LPS. huMoDCs were incubated with either RHDV-VLPs (50 μg/ml) or LPS (1 μg/ml), and after 24 h of culture, surface expression of MHC-II and CD86 was determined by flow cytometry using PE-labelled secondary mAb. (A) Dot-plot shows the gating strategy based on FSC/SSC. R0 region indicates huMoDCs. (B) Histograms show expression patterns on huMoDCs gated for FS/SS. RHDV-VLP-pulsed huMoDCs (solid line) and LPS-exposed huMoDCs (dotted line) express higher levels of all markers as compared with un-stimulated poBMDCs in steady state (grey histograms) or isotype control (black histograms). Results of one representative experiment out of two independent experiments.