The protective immune response generated by a commercial monovalent inactivated vaccine against bluetongue virus serotype 1 (BTV1) was studied. Five sheep were vaccinated, boost-vaccinated, and then challenged against BTV1 ALG/2006. RT-PCR did not detect viremia at any time during the experiment. Except a temperature increase observed after the initial and boost vaccinations, no clinical signs or lesions were observed. A specific and protective antibody response checked by ELISA was induced after vaccination and boost vaccination. This specific antibody response was associated with a significant increase in B lymphocytes confirmed by flow cytometry, while significant increases were not observed in T lymphocyte subpopulations (CD4+, CD8+, and WC1+), CD25+ regulatory cells, or CD14+ monocytes. After challenge with BTV1, the antibody response was much higher than during the boost vaccination period, and it was associated with a significant increase in B lymphocytes, CD14+ monocytes, CD25+ regulatory cells, and CD8+ cytotoxic T lymphocytes.
Bluetongue virus (BTV), a member of the
Vaccination has proven very effective in BTV control and eradication strategies [
Several inactivated BTV vaccines have been shown to confer protection mainly by inducing production of neutralizing antibodies [
Evaluating the cell-mediated immunity in animals vaccinated against BTV could provide valuable information for assessing and improving the efficacy of BTV vaccines. To that end, the present study aimed to examine
All procedures were carried out in accordance with the Code of Practice for Housing and Care of Animals Used in Scientific Procedures, approved by the European Economic Community in 1986 (86/609/EEC) and amended by European Commission Directive 2003/65/EC. The procedures were also approved by the Animal Experimental Committee of Complutense University of Madrid.
Five female Merino sheep of 9-10 months old and negative for antigens or antibodies against BTV were housed in Biosafety Level 3 facilities (VISAVET, Complutense University of Madrid).
A commercial inactivated vaccine against BTV serotype 1 was used (Zulvac1, Fort Dodge Veterinaria SA). The active component per dose (2 mL) was BTV-1/ALG2006/01 E1 ≥ 106.4 TCID50. This vaccine contains adjuvants such as
Vaccination was carried out SC on day 0 of the experiment. Booster vaccination was performed by the same route on day 20. In both cases, the vaccine dose was 2 mL as recommended by manufacturer. On day 48, all animals were challenged with 1 mL of BTV1 ALG/2006 at a virus title of 1.9 × 106 TCID50 in BHK cells. The challenge inoculum contained 1.9
Rectal temperature was measured on day 0 prior to vaccination, as well as on various days until the end of the trial on day 68. On each of these occasions, clinical signs were scored using the system described by Perrin et al. [
Serum samples were collected on day 0 prior to vaccination, as well as on days 3, 14, 16, 20, 21, 23, 26, 35, 42, 48, 51, 53, 54, 57, 58, 61, 62, and 68. Samples were analyzed by a double-recognition ELISA (Ingezim BTV DR 12.BTV.K0, Ingenasa) according to the manufacturer’s instructions. Antibody response was measured as optical density.
Samples of EDTA blood were collected on day 0 prior to vaccination, as well as on days 3, 20, 21, 23, 42, 48, 51, 53, 54, 57, 58, 61, 62 and 68. RNA was extracted from the samples using the NucleoSpin RNA II kit (Macherey-Nagel). The presence of BTV RNA was assessed using real-time RT-PCR (RT-qPCR) targeting BTV segment 5. Briefly, this RT-qPCR was able to detect up to 100 RNA copies. The relationship between the Ct and the copy number was linear between 17 and 33 cycles, which correspond to 1 × 108 and 1 × 103 copies, respectively. The RT-qPCR had an efficiency of 96%, and it was associated with an R2 of 0.99. The RT-qPCR was able to detect the mRNA in all of the 128 biological samples from sheep, goats, and cattle tested [
EDTA blood samples were collected on day 0 prior to vaccination, as well as on days 3, 14, 16, 20, 21, 23, 26, 35, 42, 48, 51, 53, 54, 57, 58, 61, 62, and 68. Flow cytometry using an FACS scan cytometer (Becton Dickinson) was used to detect different populations of peripheral blood mononuclear cells (PBMCs) (Table
Antibodies used to analyze PBMC populations by flow cytometry.
Primary and secondary antibodies | Specificity | Ig isotype | Amount per tube ( | Source | Reference |
---|---|---|---|---|---|
Anti-sheep B lymphocytes | B lymphocytes | IgM | 2 | VMRD | BAQ44A |
Anti-sheep CD4 | T helper lymphocytes | IgG1 | 2 | VMRD | 17D1 |
Anti-sheep CD8 | Cytotoxic T lymphocytes | IgG1 | 2 | VMRD | CACT80C |
Anti-sheep WC1 | IgG1 | 2 | VMRD | IL-A29 | |
Anti-sheep CD25 | IL-2 receptor | IgG1 | 2 | VMRD | CACT116A |
Anti-sheep CD14 | Monocytes | IgG1 | 2 | VMRD | CAM36A |
FITC-conjugated anti-mouse IgG1 ( | Mouse IgG1 ( | — | 0.4 | Invitrogen | P-21129 |
PE-conjugated anti-mouse IgM | Mouse IgM | — | 0.4 | Sigma-Aldrich | F-9259 |
Data were analyzed using GraphPad InStat 3.0 and IBM SPSS Statistics 19. Percentages of PBMC subpopulations (CD4+, CD8+, WC1+, CD25+, B lymphocytes, and CD14+) are reported as mean ± standard deviation (SD). Differences between the percentages of PBMC populations at different times and the values prior to vaccination on day 0 were analyzed by repeated-measures ANOVA with the Huynh-Feldt correction.
Optical density results from ELISA testing of serum samples are reported as mean ± SD. Differences among mean optical density values after vaccination (days 0–20), after boost vaccination (days 21–48), and after challenge (days 51–68) were analyzed using the Mann-Whitney
For all comparisons, differences for which
Hyperthermia (rectal temperature higher than 40°C) was detected in three sheep on day 1 after vaccination and in three sheep on day 21 after boost vaccination. After challenge, however, no increase in temperature was detected in any animal.
No BTV clinical signs were observed during the experiment. Moreover, necropsy failed to detect gross lesions characteristic of BTV infection. Histopathology confirmed the absence of microscopic lesions characteristic of BTV.
A specific antibody response against BTV was detected in all vaccinated sheep from day 14 through the end of the experiment (Figures
(a) Antibody response in serum samples (mean optical density ± SD) measured by ELISA during the experiment. The threshold below which a response was considered negative was defined as 15% of the positive control optical density. Thus, samples with an optical density > 0.252 were considered positive. The mean value after boost vaccination was significantly different from that after challenge (**
Mean values of antibody response were compared for the periods after vaccination (days 0–20), after boost vaccination (days 21–48), and after challenge (days 51–68). No significant differences were observed between antibody levels after vaccination (
No viral genome was detected in the animals at any time in the study.
CD4
Mean percentages (± SD) and individual percentages of PBMC populations labeled by antibodies against CD4
The mean percentage of CD8
The percentage of
The percentage of CD25
Mean percentages (± SD) and individual percentages of PBMC populations labeled by antibodies against CD25
The percentage of B cells decreased significantly after vaccination, with the lowest level of 15.4% occurring on day 14 (Figures
The percentage of monocytes labeled by the anti-CD14
The inactivated vaccine against BTV serotype 1 (Zulvac1, Fort Dodge Veterinaria SA) induced an effective immune response in all vaccinated sheep. Challenge virus was not detected in blood by RT-PCR even at 20 days after inoculation. Except for a temperature increase observed in most animals after the vaccination and boost vaccination, no clinical signs or lesions characteristic of BTV infection were observed.
The temperature increase observed in most animals after vaccination and boost vaccination may reflect activation/stimulation of the immune system. Similar increases were observed after administration of inactivated vaccines against different serotypes of BTV [
After subcutaneous administration of inactivated vaccines against BTV, the presence of viral RNA in blood should not be detected [
At 14 days after vaccination, a specific antibody response against BTV was observed in all vaccinated sheep, and it persisted through the end of the experiment. This antibody response was shown to be the main factor in protecting sheep against BTV serotype 1. Our results are in agreement with previous studies [
The potential role of the cellular immune response during BTV infection and after vaccination is not fully understood [
The lack of cell-mediated immunity after vaccination and boost vaccination in our experiment could be attributed to different components of the vaccine, in which inactivated virus was mixed with an aluminum-based adjuvant. These adjuvants delay the elimination of antigens after vaccine administration, prolonging the antigenic stimulus. These adjuvants also promote antibody response, even though they have little stimulatory effect on cell-mediated responses [
On the other hand, B cells can recognize most antigens without prior processing, and certain antigens can provoke antibody formation in the absence of helper T cells, providing sufficient signal for B cell proliferation and differentiation into antibody-producing plasma cells [
After challenge, viral genome was not detected in vaccinated sheep, confirming specific protection induced by antibody response. In fact, antibody response after challenge was significantly higher than after boost vaccination, and this increase was associated with a significant increase in CD14
Finally, after challenge with BTV serotype 1, an increase in CD8
Collectively, these results demonstrate that a specific and protective antibody response was induced after vaccination and boost vaccination of sheep with an inactivated vaccine against BTV serotype 1 (Zulvac1, Fort Dodge Veterinaria SA). This response was associated with a significant increase in B lymphocytes. However, significant increases were not observed in T lymphocyte subpopulations (CD4
None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
This study was funded by projects BTVAC FP6-2005-SSP-5A and AGL2009-13174-C02-01. A. C. Pérez de Diego holds a scholarship of the FPU programme (Ministry of Education and Science, Spain). P. J. Sánchez-Cordón is supported by a contract of the “Ramón y Cajal programme” (Ministry of Science and Innovation, Spain). The authors thank Amalia for assistance during flow cytometry analysis. They also thank Rocío Sánchez, Belén Rivera, and Pablo del Carmen for dedicating their time and effort to this study.