Characterization of protection afforded by a bivalent virus-like particle vaccine against bluetongue virus serotypes 1 and 4 in sheep

Abstract

Background: Bluetongue virus (BTV) is an economically important, arthropod borne, emerging pathogen in Europe, causing disease mainly in sheep and cattle. Routine vaccination for bluetongue would require the ability to distinguish between vaccinated and infected individuals (DIVA). Current vaccines are effective but are not DIVA. Virus-like particles (VLPs) are highly immunogenic structural mimics of virus particles, that only contain a subset of the proteins present in a natural infection. VLPs therefore offer the potential for the development of DIVA compatible bluetongue vaccines.

Methodology/principal findings: Merino sheep were vaccinated with either monovalent BTV-1 VLPs or a bivalent mixture of BTV-1 VLPs and BTV-4 VLPs, and challenged with virulent BTV-1 or BTV-4. Animals were monitored for clinical signs, antibody responses, and viral RNA. 19/20 animals vaccinated with BTV-1 VLPs either alone or in combination with BTV-4 VLPs developed neutralizing antibodies to BTV-1, and group specific antibodies to BTV VP7. The one animal that showed no detectable neutralizing antibodies, or group specific antibodies, had detectable viral RNA following challenge but did not display any clinical signs on challenge with virulent BTV-1. In contrast, all control animals' demonstrated classical clinical signs for bluetongue on challenge with the same virus. Six animals were vaccinated with bivalent vaccine and challenged with virulent BTV-4, two of these animals had detectable viral levels of viral RNA, and one of these showed clinical signs consistent with BTV infection and died.

Conclusions: There is good evidence that BTV-1 VLPs delivered as monovalent or bivalent immunogen protect from bluetongue disease on challenge with virulent BTV-1. However, it is possible that there is some interference in protective response for BTV-4 in the bivalent BTV-1 and BTV-4 VLP vaccine. This raises the question of whether all combinations of bivalent BTV vaccines are possible, or if immunodominance of particular serotypes could interfere with vaccine efficacy.

Figure 1. Group specific immune response of sheep vaccinated with VLPs.

Sero-conversion of vaccinated sheep was monitored using competitive ELISA (POURQUIER® Bluetongue Competitive ELISA) specific to VP7, the BTV group specific core antigen. V1 and V2 indicate the day sheep were vaccinated and boosted with immunogen, respectively. C indicates the day sheep were challenged with virulent BTV. The data represented is the average of all animals in each group, no adjustments or omissions have been made. The threshold for the sample to be considered as seropositive for VP7 was 35%. A. ELISA results of groups of animals were immunised with VLPs and challenged with virulent BTV-1. All vaccinated animals, except A2 had a BTV group specific antigen response above threshold as determined by the kit prior to challenge. None of the control animals had a response prior to challenge. Average results of groups of sheep vaccinated with different immunogens are shown: Group D (control:▴), Group A (BTV-1 VLPs:⧫) and Group B (BTV-1 & BTV-4 VLPs:□). B. ELISA results of groups of animals vaccinated with VLPs and challenged with virulent BTV-4. All vaccinated animals had a BTV group specific antigen response prior to challenge. None of the control animals had a response prior to challenge. Average results of groups of sheep vaccinated with different immunogens are shown: Group E, (saline+adjuvant: ▪) and Group C (BTV-1 VLPs & BTV-4 VLPs: •).

Figure 2. Neutralisation antibody response of sheep vaccinated with VLPs.

Virus neutralisation antibody titres of individual sheep vaccinated with BTV-1 VLP (black), BTV-1 & BTV-4 VLP (grey), BTV-1 & BTV-4 VLP (white) and non-vaccinated sheep (black and white striped or spotted bars) prior to challenge with virulent virus as determined by the neutralisation of 50 TCID₅₀. The group of each animal is indicated by an A, B, C, D or E alongside the animal number. The serum neutralisation titres against BTV-4 were not determined for animals A6 (Group A) and C5 (Group C) due to lack of serum collected. A. Neutralisation antibodies elicited against BTV-1. B. Neutralisation antibodies elicited against BTV-4. Titres below the threshold of the assay (dashed line) were considered to be negative.

Figure 3. The average clinical score of sheep challenged with virulent BTV-1 (A) or BTV-4 (B).

All sheep vaccinated with BTV-1 VLPs (Group A; black bar), BTV-1 & BTV-4 VLP (Group B; grey bar) and challenged with virulent BTV-1 were protected from detectable disease. All control sheep challenged with BTV-1 (Group D; striped bar) developed clinical signs of bluetongue disease. Sheep vaccinated with BTV-1 and BTV-4 VLPs (Group C; white bar) and challenged with BTV-4 show some clinical score on this chart because of one animal (C5). No other animal in the group showed clinical signs of bluetongue. The control animals challenged with BTV-4 (Group E; spotted bars) developed mild clinical bluetongue. Individual sheep were scored on a standard, 8 point clinical reaction scale. Data presented in the chart is the average score for each group.

Figure 4. Temperature response of sheep challenged with virulent BTV.

The average rectal temperatures (°C) of BTV-1 VLP (Group A) and BTV-1 & BTV -4 VLP (Group B and C) vaccinated and saline vaccinated sheep (Group D and E) were monitored after challenge. The time of challenge is indicated by an arrow. All animals in each group were included in the analysis. A. Animals challenged with virulent BTV-1. The different groups are indicated; Group D (▴), Group A (⧫) and Group B (□). B. Animals challenged with virulent BTV-4. The different groups are indicated; Group E (▪) and Group C (•).

Figure 5. Detection of viraemia in vaccinated animals following challenge with virulent BTV.

BTV genomic RNA was detected in blood samples by quantitative RT-PCR follow throughout the experiment. None of the animals had any detectable viral RNA prior to challenge. Each Ct values represented is the average of all animals in each group, no adjustments or omissions have been made. A. Animals were challenged with virulent BTV-1. Post- challenge, none of the sheep vaccinated with bivalent BTV-1 & BTV-4 VLPs vaccine (Group B) had detectable viraemia, one sheep vaccinated with monovalent BTV-1 VLPs (Group A) had detectable viraemia and all saline vaccinated control animals (Group D) had detectable viraemia. The different groups are indicated; Group D (▴), Group A (⧫) and Group B (□). B. Animals were challenged with virulent BTV-4. All control animals (Group E) developed viraemia, while 2 of the 6 animals vaccinated with bivalent BTV-1 & BTV-4 VLPs vaccine (Group C) had detectable viral dsRNA. The different groups are indicated; Group E (▪) and Group C (•).