Long-term persistence of neutralising antibodies against bluetongue virus serotype 8 in naturally infected cattle

Neutralising antibodies to bluetongue virus (BTV) in convalescent cattle have been described as persistent. Controlled laboratory studies, however, rarely last longer than a couple of weeks and long-term field data are lacking. This study followed twelve cattle that had been naturally infected with bluetongue virus serotype 8 (BTV-8) in Germany in 2006. Using ELISAs and a serum neutralisation test, we found a strong humoral immune response four to six years after the last exposure to BTV-8; based on data from long-term vaccine studies, it is highly likely that this coincides with immunity to reinfection with the same serotype.     

Seroconversion,neutralising antibodies and protection in bluetongue serotype 8 vaccinated sheep

Bluetongue virus serotype 8 (BTV-8) has caused a major outbreak of disease in cattle and sheep in several countries across northern and western Europe from 2006 to 2008. In 2008 the European Union instigated a mass-vaccination programme in affected countries using whole virus inactivated vaccines. We evaluated vaccinal responses in sheep and the ability of the vaccine to protect against experimental challenge. Sheep vaccinated 10 months previously under field conditions were challenged with BTV-8. One of 7 vaccinated sheep became infected, as evidenced by detection of viral RNA by real-time RT-PCR and by virus isolation. The remaining 6 sheep appeared fully protected from virus replication. None of the vaccinated sheep showed clinical signs of BTV and there was a good correlation between the presence of neutralising antibodies on challenge and protection. Commercially available ELISAs were evaluated for their ability to detect antibodies in sheep vaccinated on a single occasion. The sandwich (double antigen) ELISA assays were found to be more sensitive at detecting antibodies in vaccinated sheep than the competitive ELISAs.     

Two alternative inocula to reproduce bluetongue virus serotype 8 disease in calves

The aim of this study was to investigate the consequences in calves of two forms of inocula alternative to the use of wild type infectious blood. Two groups of five calves were infected with low cell-passaged virus and infectious blood issued from one animal passage of the same strain. A longitudinal study was implemented and characterised by clinical standardised observations, haematology, BTV RNA detection and viral isolation from blood, detection of serogroup and neutralising antibodies, cytokine expression and post-mortem examination 46 days post-infection (PI). Both tested inocula were able to reproduce clinical expression of the disease, in the bloodstream viral genome was detected until the end of the experiment while virus isolation was possible between days 7 and 31 PI. Humoral immune response developed earlier in calves infected with low cell-passaged virus, while in both groups a massive antibody production was confirmed by the immune balance between IL-4 and IFN-γ expression. Both tested inocula are presented as valid alternative to the use of wild type infectious blood in the study of the pathogenesis of BTV-8 or the efficacy of current and future vaccines.     

VP2-serotyped live-attenuated bluetongue virus without NS3/NS3a expression provides serotype-specific protection and enables DIVA

Bluetongue virus (BTV) causes Bluetongue in ruminants and is transmitted by Culicoides biting midges. Vaccination is the most effective measure to control vector borne diseases; however, there are 26 known BTV serotypes showing little cross protection. The BTV serotype is mainly determined by genome segment 2 encoding the VP2 protein. Currently, inactivated and live-attenuated Bluetongue vaccines are available for a limited number of serotypes, but each of these have their specific disadvantages, including the inability to differentiate infected from vaccinated animals (DIVA).BTV non-structural proteins NS3 and NS3a are not essential for virus replication in vitro, but are important for cytopathogenic effect in mammalian cells and for virus release from insect cells in vitro. Recently, we have shown that virulent BTV8 without NS3/NS3a is non-virulent and viremia in sheep is strongly reduced, whereas local in vivo replication leads to seroconversion. Live-attenuated BTV6 without NS3/NS3a expression protected sheep against BTV challenge. Altogether, NS3/NS3a knockout BTV6 is a promising vaccine candidate and has been named Disabled Infectious Single Animal (DISA) vaccine.Here, we show serotype-specific protection in sheep by DISA vaccine in which only genome segment 2 of serotype 8 was exchanged. Similarly, DISA vaccines against other serotypes could be developed, by exchange of only segment 2, and could therefore safely be combined in multi-serotype cocktail vaccines with respect to reassortment between vaccine viruses.Additionally, NS3 antibody responses are raised after natural BTV infection and NS3-based ELISAs are therefore appropriate tools for DIVA testing accompanying the DISA vaccine. To enable DIVA, we developed an experimental NS3 ELISA. Indeed, vaccinated sheep remained negative for NS3 antibodies, whereas seroconversion for NS3 antibodies was associated with viremia after heterologous BTV challenge.     

Evaluation of the humoral immune response in adult dairy cattle three years after vaccination with a bluetongue serotype 8 inactivated vaccine

Despite the widespread use of bluetongue serotype 8 (BTV-8) inactivated vaccines across Europe from 2008 to 2011, two very practical questions remain unanswered about the length of persistence of group-specific antibodies in milk and serum post-vaccination and the duration of protection beyond one year post-vaccination. This study has firstly revealed that group-specific antibodies persist at high levels in milk and serum in the majority of cattle for at least 3 years post-vaccination, thus removing the option of using these animals in ELISA-based surveillance programmes. Secondly neutralising antibodies have been shown to persist in the majority of cattle for at least 3 years post-vaccination, indicating that the cattle are likely to be protected for this time period. This extended duration of protection may have contributed towards the rapid and efficient eradication of BTV-8 from many European countries, despite reducing levels of vaccine coverage.     

Evaluation of humoral response and protective efficacy of three inactivated vaccines against bluetongue virus serotype 8 one year after vaccination of sheep and cattle

The long-term efficacy of three commercially available inactivated vaccines against bluetongue virus serotype 8 (BTV-8) (BLUEVAC^® 8, Zulvac^® 8, and BTVPUR^® AlSap 8) was evaluated in a seroprevalence study and challenge experiments. Seroprevalences 1 year after vaccination ranged from 75% to 100%. In two infection experiments, groups of vaccinated sheep and cattle selected either randomly or for low antibody levels were challenged with a European BTV-8 strain 12 months after vaccination. With two exceptions, all animals, including those with low antibody levels prior to challenge, were protected from viral replication and clinical disease even at low initial antibody levels. Vaccination of susceptible ruminants in yearly intervals is thus considered an adequate scheme for BTV-8 control in Europe.     

Rift Valley fever virus subunit vaccines confer complete protection against a lethal virus challenge

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus causing significant morbidity and mortality in livestock and humans. Rift Valley fever is endemic in Africa, but also outside this continent outbreaks have been reported. Here we report the evaluation of two vaccine candidates based on the viral Gn and Gc envelope glycoproteins, both produced in a Drosophila insect cell expression system. Virus-like particles (VLPs) were generated by merely expressing the Gn and Gc glycoproteins. In addition, a soluble form of the Gn ectodomain was expressed and affinity-purified from the insect cell culture supernatant. Both vaccine candidates fully protected mice from a lethal challenge with RVFV. Importantly, absence of the nucleocapsid protein in either vaccine candidate facilitates the differentiation between infected and vaccinated animals using a commercial recombinant nucleocapsid protein-based indirect ELISA.     

Clinical disease in sheep caused by bluetongue virus serotype 8, and prevention by an inactivated vaccine

The ability to reduce clinical signs, induce neutralizing antibodies, and perhaps most importantly, to prevent or reduce viraemia (and therefore virus-transmission), represent primary criteria for assessment of bluetongue virus (BTV) vaccine efficacy. Identification of BTV challenge-strains that reliably induce viraemia and clinical signs comparable to those in naturally infected animals, is therefore important for vaccine evaluation. Texel cross-breed and Dorset Poll sheep vaccinated with inactivated BTV-8 vaccine (‘Bovilis^® BTV8’ from MSD Animal Health), were challenged with low-passage BTV-8 (Northern European strain) grown in either insect (Culicoides) or mammalian cell-cultures. The severity of clinical signs was recorded (using a modified numerical scoring-system, which is described) along with viraemia and serum neutralizing (SN) antibody levels. Low level SN-antibodies were detected at the time of challenge (three weeks after vaccination). All unvaccinated control animals became infected after challenge, developing high SN-antibody titres by 21 days post challenge (dpc). Vaccinees showed faster increases in SN-antibody titres (‘booster’ response), with significantly higher titres at 6 dpc than unvaccinated controls. Although only limited clinical-signs could be attributed to BTV in younger animals infected with the mammalian-cell-culture derived virus, both BTV-8 challenge preparations induced severe clinical signs comparable to ‘bluetongue’ observed during natural outbreaks in older unvaccinated animals. Challenge with BTV-8 grown in Culicoides cell-cultures seemed to induce greater severity of clinical-scores and ‘post-mortem lesions’ than the mammalian-derived BTV-8 strain. Vaccination reduced clinical signs, fever, and viraemia equally well after challenge with either virus preparation.     

Evaluation of the humoral immune responses in adult cattle and sheep, 4 and 2.5 years post-vaccination with a bluetongue serotype 8 inactivated vaccine

One of the big surprises about the devastating outbreak of bluetongue serotype-8 that spread across Northern and Western Europe between 2006 and 2008 was how relatively quickly the virus was controlled and eradicated from affected countries. This was at least in part attributed to the high levels of vaccine coverage achieved in affected countries. A previous study revealed that neutralising antibodies persisted in the majority of vaccinated cattle for at least 3 years post-vaccination, indicating that cattle are likely to be protected for this time period. The current study revealed that neutralising antibodies persisted in the same group of cattle for up to 4 years post-vaccination, and that neutralising antibodies persisted for up to 2.5 years in sheep that had been vaccinated on two occasions one year apart. These results have implications for future bluetongue surveillance programmes and vaccine control strategies.     

Efficacy of three inactivated vaccines against bluetongue virus serotype 8 in sheep

Bluetongue has become a major animal health issue in the European Union. The member states and Switzerland have agreed on a vaccination strategy. Three different inactivated monovalent vaccines against bluetongue virus serotype 8 were selected for the compulsory vaccination program carried out in Germany in 2008. The efficacy of these vaccines was evaluated in a pilot study in sheep immunised under field conditions by clinical, virological and serological examination before and after experimental challenge infection with a BTV-8 field isolate. Antibody levels prior to challenge infection differed between the vaccinated groups, but all seroconverted animals were fully protected against clinical disease and virus replication. Only one vaccinated animal was very weakly positive in the real-time RT-PCR at day 10 after challenge infection, and one seronegative sheep in one of the vaccine groups was not protected.     

Protective duration of immunity of an inactivated bluetongue (BTV) serotype 2 vaccine against a virulent BTV serotype 2 challenge in sheep

The protective properties of an inactivated bluetongue virus serotype 2 (BTV-2) vaccine were evaluated in sheep. Sheep (two groups of seven), vaccinated with either one or two doses of the vaccine, were monitored for antibody response over one year. All sheep developed high titres of neutralizing antibodies by 35 days after first vaccination and titres were maintained over one year. Control sheep (n = 7) remained seronegative until challenge. One year after vaccination, all sheep were inoculated with a virulent BTV-2. All controls developed pyrexia, clinical signs and viraemia. In contrast, the sheep vaccinated with one or two doses of inactivated BTV-2 vaccine were protected from clinical disease and viraemia was completely prevented. These data show that a single dose of the BTV-2 vaccine given to sheep induces a strong immunity which confers protection for at least one year.     

Molecular epidemiology of bluetongue virus serotype 1 circulating in Italy and its connection with northern Africa

Western BTV-1 emerged in the Mediterranean basin in 2006 and it has since been isolated in southern and northern European countries. Six BTV-1 strains isolated from infected sheep in Italy between 2006 and 2013 and a BTV-1 strain isolated from an infected sheep in Tunisia in 2011 were fully sequenced. The seven strains were shown to be nearly identical in each gene segment. The Seg-2 sequences of the BTV-1 strains group according to the year of isolation reflecting the time of BTV incursions in Italy. Combined results suggest that BTV-1 strains isolated in Sardinia, Sicily and mainland Italy in 2012 and 2013 have a direct northern African origin. The Italian strains originated from a strain closely related to a BTV-1 strain isolated in Tunisia in 2011. Better surveillance programs with northern and sub-Saharan African countries should be implemented making the control of spread of BTV easier and effective.     

Effect of vaccination with an inactivated vaccine on transplacental transmission of BTV-8 in mid term pregnant ewes and heifers

The effect of vaccination with a commercial inactivated Bluetongue virus serotype 8 (BTV-8) vaccine on the ability of BTV-8 to cross the ruminant placenta was investigated in two experiments. Ten pregnant ewes (Experiment 1) or heifers (Experiment 2) were vaccinated according to the manufacturer's instructions. Three weeks after the completion of the vaccination schedule, all vaccinated animals were infected with BTV-8 together with ten non-vaccinated pregnant animals that served as challenged controls. Four additional pregnant animals received a mock challenge at the same time point. Three weeks after the challenge, the foetuses were collected. In the sheep experiment, the lambs of the vaccinated ewes and the mock infected ewes were negative in the virus isolation, whereas BTV-8 could be isolated from 11/23 lambs of 6/10 ewes in the BTV-8 challenged control group. The incidence and severity of BTV associated lesions, such as haemorrhages, meningitis/encephalitis and necrosis in the placentomes was significantly higher in the BTV-8 challenged control group. The rate of transplacental transmission was less in the cattle experiment: BTV-8 could be detected in 2/10 calves in the BTV-8 challenged control group. All other calves were negative.Vaccination clearly reduced transplacental transmission of BTV-8 in the sheep experiment, whereas in the cattle experiment, the incidence of transmission was too low to demonstrate a significant reduction of transmission by vaccination. However, the vaccine very effectively blocked viraemia, which suggests that the vaccine might prevent transmission in cattle as well.Transplacental transmission of BTV has serious economical consequences, due to the loss of progeny to the livestock industry. Vaccination can be an important aid in the reduction of these economic losses.     

Bluetongue virus serotype 8 reemergence in Germany, 2007 and 2008

Reemerging bluetongue virus serotype 8 (BTV-8) in Germany was detected first in May 2007 in a sentinel cow and in February 2008 in an export heifer. Reemergence was confirmed by retesting the samples, experimental inoculation, fingerprinting analysis, and virus isolation. Overwintering of BTV-8 and continuous low-level infections are assumed.     

An experimental vaccine composed of two adjuvants gives protection against Mycoplasma bovis in calves

Mycoplasma bovis is a major pathogen affecting cattle causing bronchopneumonia, mastitis, and other disorders. Only autogenous vaccines made specifically for individual farms are available in parts of Europe and the United States. A novel experimental vaccine composed of a field M. bovis isolate combined with saponin and Emulsigen^® adjuvants was evaluated. Eighteen 3–4 week old calves were placed in three equal groups: vaccinated (Vac), positive control (PC) and negative control (NC). The Vac calves were subcutaneously injected with 8 ml of the vaccine; the PC and NC calves received phosphate buffered saline (PBS). Three weeks later the Vac and PC calves were challenged with a virulent M. bovis strain, the NC group received PBS. Throughout the study clinical observations, microbiology and immunological tests were carried out. Three weeks post challenge two calves from each group were euthanased for necropsy and histopathological examination. The vaccine effectively stimulated the humoral immune response, with high titres of anti-M. bovis specific antibodies and total Ig concentration. This vaccine also intensified the IgA response. A clinically protective effect of the vaccine was demonstrated as it also reduced the gross pathological lung lesions and nasal shedding of M. bovis.     

Evaluation of adaptive immune responses and heterologous protection induced by inactivated bluetongue virus vaccines

Eradication of bluetongue virus is possible, as has been shown in several European countries. New serotypes have emerged, however, for which there are no specific commercial vaccines. This study addressed whether heterologous vaccines would help protect against 2 serotypes. Thirty-seven sheep were randomly allocated to 7 groups of 5 or 6 animals. Four groups were vaccinated with commercial vaccines against BTV strains 2, 4, and 9. A fifth positive control group was given a vaccine against BTV-8. The other 2 groups were unvaccinated controls. Sheep were then challenged by subcutaneous injection of either BTV-16 (2 groups) or BTV-8 (5 groups). Taken together, 24/25 sheep from the 4 experimental groups developed detectable antibodies against the vaccinated viruses. Furthermore, sheep that received heterologous vaccines showed significantly reduced viraemia and clinical scores for BTV-16 when compared to unvaccinated controls. Reductions in clinical signs and viraemia among heterologously vaccinated sheep were not as common after challenge with BTV-8. This study shows that heterologous protection can occur, but that it is difficult to predict if partial or complete protection will be achieved following inactivated-BTV vaccination.     

The development of recombinant subunit envelope-based vaccines to protect against dengue virus induced disease

Challenges associated with the interference observed between the dengue virus components within early tetravalent live-attenuated vaccines led many groups to explore the development of recombinant subunit based vaccines. Initial efforts in the field were hampered by low yields and/or improper folding, but the use of the Drosophila S2 cell expression system provided a mechanism to overcome these limitations. The truncated dengue envelope proteins (DEN-80E) for all four dengue virus types are expressed in the S2 system at high levels and have been shown to maintain native-like conformation. The DEN-80E proteins are potent immunogens when formulated with a variety of adjuvants, inducing high titer virus neutralizing antibody responses and demonstrating protection in both mouse and non-human primate models. Tetravalent vaccine formulations have shown no evidence of immune interference between the four DEN-80E antigens in preclinical models. Based on the promising preclinical data, the recombinant DEN-80E proteins have now advanced into clinical studies. An overview of the relevant preclinical data for these recombinant proteins is presented in this review.     

Biopolymer encapsulated live influenza virus as a universal CD8+ T cell vaccine against influenza virus

Current influenza virus vaccines primarily elicit antibodies and can be rendered ineffective by antigenic drift and shift. Vaccines that elicit CD8+ T cell responses targeting less variable proteins may function as universal vaccines that have broad reactivity against different influenza virus strains. To generate such a universal vaccine, we encapsulated live influenza virus in a biopolymer and delivered it to mice subcutaneously. This vaccine was safe, induced potent CD8+ T cell immunity and protected mice against heterosubtypic lethal challenge. Safety of subcutaneous (SQ) vaccination was tested in Rag−/−γc−/− double knockout mice which we show cannot control intranasal infection. Biopolymer encapsulation of live influenza virus could be used to develop universal CD8+ T cell vaccines against heterosubtypic and pandemic strains.