Purification, stability, and immunogenicity analyses of five bluetongue virus proteins for use in development of a subunit vaccine that allows differentiation of infected from vaccinated animals

Abstract

Bluetongue virus (BTV) causes bluetongue disease, a vector-borne disease of ruminants. The recent northerly spread of BTV serotype 8 in Europe resulted in outbreaks characterized by clinical signs in cattle, including unusual teratogenic effects. Vaccination has been shown to be crucial for controlling the spread of vector-borne diseases such as BTV. With the aim of developing a novel subunit vaccine targeting BTV-8 that allows differentiation of infected from vaccinated animals, five His-tagged recombinant proteins, VP2 and VP5 of BTV-8 and NS1, NS2, and NS3 of BTV-2, were expressed in baculovirus or Escherichia coli expression systems for further study. Optimized purification protocols were determined for VP2, NS1, NS2, and NS3, which remained stable for detection for at least 560 to 610 days of storage at +4°C or -80°C, and Western blotting using sera from vaccinated or experimentally infected cattle indicated that VP2 and NS2 were recognized by BTV-specific antibodies. To characterize murine immune responses to the four proteins, mice were subcutaneously immunized twice at a 4-week interval with one of three protein combinations plus immunostimulating complex ISCOM-Matrix adjuvant or with ISCOM-Matrix alone (n = 6 per group). Significantly higher serum IgG antibody titers specific for VP2 and NS2 were detected in immunized mice than were detected in controls. VP2, NS1, and NS2 but not NS3 induced specific lymphocyte proliferative responses upon restimulation of spleen cells from immunized mice. The data suggest that these recombinant purified proteins, VP2, NS1, and NS2, could be an important part of a novel vaccine design against BTV-8.

FIG 1

Optimization process of purification protocols for recombinant BTV proteins. Variations in the buffers were tried in sequence as indicated horizontally, until acceptable purity was achieved.

FIG 2

Degree of purification and presence of recombinant His-tagged NS3 of BTV-2. Coomassie-stained SDS-PAGE (A) or Western blot using mouse antihistidine monoclonal antibodies (B) showing the degree of purification and presence of NS3 after purification using His SpinTrap columns with different adjustments of lysis buffer contents: following the manufacturer's protocols (lane 1), adding 2% octyl glucoside (lane 2), increasing the salt concentration 5× (lane 3), decreasing the salt concentration 10× (lane 4), and using NP-40 lysis buffer (lane 5, optimized lysis buffer). Semiquantitive purity percentages are indicated below each lane in panel A. Arrowheads indicate recombinant BTV proteins at expected molecular masses. Molecular mass markers (in kDa) are noted at the right of each image.

FIG 3

Expression and purification of recombinant VP2 and VP5 from BTV-8 and NS1, NS2, and NS3 from BTV-2. A Coomassie-stained SDS-PAGE gel (A) or Western blot using mouse antihistidine monoclonal antibodies (B) confirming expression and purification of recombinant His-tagged VP2, VP5, NS1, and NS3 from baculovirus-infected Sf9 cells and NS2 from transformed E. coli BL21-AI is shown. Recombinant proteins were purified using cobalt or nickel affinity. Semiquantitive purity percentages are indicated below each lane in panel A. Arrowheads indicate recombinant BTV proteins at expected molecular masses. Molecular mass markers (in kDa) are noted at the right of each image.

FIG 4

Kinetics of protein-specific serum antibody titers directed against VP2 of BTV-8 (A) or NS2 of BTV-2 (B) in immunized mice. Mice (represented by group means of log₁₀-transformed values) were immunized twice at a 4-week interval with one of three combinations of purified recombinant BTV proteins and AbISCO-100 (abbreviated as vVP2 [n = 5], vNS1/2/3 [n = 6], or vVP2NS1/2/3 [n = 6]) or with AbISCO-100 alone (Control; n = 6). Sera were collected 4 and 2 weeks after the first and second vaccinations (indicated by arrows), respectively, and analyzed by indirect ELISA. Lines between data points are included for illustrative purposes. Standard deviations are shown by upward deflection lines. Statistical significance is indicated by asterisks (P ≤ 0.05 [*] or P ≤ 0.01 [**]) and the corresponding group names.

FIG 5

Proliferative responses of spleen lymphocytes of immunized mice stimulated ex vivo with VP2 of BTV-8 (A), NS1 of BTV-2 (B), or NS2 of BTV-2 (C). Mice were immunized as indicated in Fig. 4. Proliferation is expressed as corrected OD values (mean of quadruplicates) after 5 days of stimulation with VP2, NS1, NS2, or control antigen and addition of the alamarBlue reagent. Dots represent individual mice, and horizontal lines represent group means. Statistical significance is indicated by asterisks (P ≤ 0.05 [*] or P ≤ 0.01 [**]) and horizontal brackets between corresponding groups.

FIG 6

Western blot indicating specific recognition of purified recombinant VP2 of BTV-8 and NS2 of BTV-2 by serum antibodies induced by BTV-8 vaccination or infection. Western blots using mouse anti-histidine tag monoclonal antibodies (lane 1), murine sera after two immunizations with vVP2NS1/2/3 (lane 2), and bovine serum after two immunizations with a commercial inactivated vaccine against BTV-8 (lane 3), eight immunizations with commercial inactivated vaccines against BTV-8 (lane 4), and 3 weeks after experimental infection with BTV-8 (lane 5) are shown. Solid arrowheads indicate VP2 (111 kDa), and white arrowheads indicate NS2 (40 kDa).