The MVA-VP2-NS1-2A-NS2-Nt vaccine candidate provides heterologous protection in sheep against bluetongue virus

Abstract

Bluetongue (BT) is an important arthropod-borne livestock disease transmitted by Culicoides midges. The etiological agent, Bluetongue virus (BTV), can lead to severe economic losses due to reduced productivity and trade restrictions. Nowadays, classical vaccines based on inactivated viruses are used to control outbreaks but do not confer multiserotype protection, which reinforces the idea of pursuing research into developing strategies that enhance the immune response directed to conserved antigenic regions, aiming broader protection across multiple serotypes. Recently, we described a vaccine candidate that confers full protection against a homologous serotype of BTV based on recombinant Modified Vaccinia Virus Ankara (MVA) co-expressing the highly conserved BTV nonstructural protein NS1 and the N-terminal end of NS2 along with protein VP2 of BTV-4. In this work, we evaluated the multiserotype protective capacity of this recombinant vaccine candidate in sheep after infection with the heterologous virus BTV-8, achieving a significant blockade of viral replication and attenuation of the clinical signs induced by BTV. After infection, vaccinated animals showed more regulated pro-inflammatory cytokine levels compared to non-vaccinated sheep. In addition, we noticed the induction of potent T cell immune responses specific to NS1 and NS2-Nt proteins of BTV, mainly based on CD8+ T cells, which could mediate the protection against BTV-8. Moreover, stimulated immunized sheep PBMCs with BTV antigens triggered the secretion of IL-6, IL-1β, IL-1α, IL-17a, IL-10 and IFN-γ, cytokines that play crucial roles in initiating immune responses.

Keywords: DIVA; MVA; bluetongue virus (BTV); multiserotype; orbivirus; sheep; vaccine.

Figure 1

Protection of immunized sheep against a heterologous virulent challenge with BTV-8. (A) A group of sheep (n=4) was immunized following a homologous prime-boost regimen consisting of two doses of MVA-VP2-NS1-2A-NS2-Nt. A second group was left untreated (Control). Immunized and non-immunized sheep were challenged with BTV-8. (B) Titers of BTV-8 recovered in blood of sheep after viral inoculation. Points represent individual PFU/ml values and lines represent mean Log PFU/ml of each group. (C) RNAemia analyzed by RT-qPCR of non-immunized and immunized sheep after viral challenge. Presence of virus in blood and expression of mRNA of segment 5 (encoding NS1 protein) was quantified. Results were expressed as Ct (left y-axis). The real-time RT-qPCR specific for BTV segment 5 was performed as described by Toussaint et al. (28). Cut-off Ct ≥ 38 (dotted black line). Points represent individual Ct values and lines represent mean Ct of each group. *P value  <  0.05, **P value  <  0.002, ***P value  <  0.001, ****P value  <  0.0001 using two-way ANOVA (post hoc Tukey test for multiple comparisons).

Figure 2

Changes in rectal temperatures and hematologic parameters in immunized and non-immunized sheep after BTV-8 challenge. (A) Rectal temperatures recorded before and after challenge. The day of challenge (0 d.p.i.) is indicated. Points represent mean rectal temperature value for each group and error bars represent SD. (B, C) Percentages of lymphocytes and neutrophils in blood from immunized sheep after challenge with BTV-8. Blood of non-immunized and immunized sheep were analyzed in an autohematology analyzer (BC-5300 Vet; Mindray, China) and the percentage of lymphocytes (B) and neutrophils (C) based on the total white blood cells were analyzed at days 0, 3, 5, 7, 10 and 12 post-infection. Points indicate the individual value of each animal and lines represent the mean value of each group. No statistical differences were found by two-way ANOVA (post hoc Tukey test for multiple comparisons).

Figure 3

Levels of cytokines in serum samples from non-immunized and immunized sheep after challenge with BTV-8. Sheep sera were collected at 3 and 5 d.p.i. and analyzed in Luminex immunoas-says. Points represent individual values for each sheep, bars represent mean values of each group, and error bars represent SD. Asterisks denote significant differences between immunized and non-immunized control. * P value <  0.05, using two-way ANOVA (post hoc Tukey test for multiple comparisons).

Figure 4

Neutralizing antibodies titers against (A) BTV-4M and (B) BTV-8 in immunized and non-immunized sheep by PRNT50 assay. NAbs titers were measured in sera collected at 3 w.p.b. and 18 d.p.i. after challenge with BTV-8. Points represent individual values for each sheep, bars represent the mean values of each group, points indicate the mean value of each group and error bars represent SD. **P value < 0.005, using two-way ANOVA (post hoc Tukey test for multiple comparisons).

Figure 5

Cellular immune responses against BTV in MVA-VP2-NS1-2A-NS2-Nt immunized sheep. (A) ELISpot assay measuring IFN-γ-secreting cells after isolation of PBMCs of immunized and non-immunized sheep and stimulation with a pool of peptides of NS1, a recombinant protein NS2-Nt and an irrelevant peptide (neg). (B–D) Flow cytometry analysis. Percentage of CD4+IFN-γ+ (B), CD8+ IFN-γ+ (C) and WC+IL-2+ (D) cells after stimulation of sheep PBMCs with a pool of peptides of NS1, a recombinant protein NS2-Nt and an irrelevant peptide. Points represent individual values for each sheep, bars represent mean values of each group and error bars represent the SD. *P value < 0.05, P value < 0.002, *P value < 0.0002 using two-way ANOVA (post hoc Tukey test for multiple comparisons). Neg: negative, irrelevant peptide from Gn RVFV.

Figure 6

Secretion of cytokines after restimulation of PBMCs with NS1 peptides, NS2-Nt recombinant protein or an irrelevant antigen (Gn RVFV). Level of proteins (pg/ml) in cell supernatants at 18 h post incubation was analyzed by Luminex Multiplex assays. Points represent individual values for each sheep, bars represent mean values of each group and error bars represent the SD. *P value <  0.05, using two-way ANOVA (post hoc Tukey test for multiple comparisons) ** P value < 0.005.