Utrecht University (logo) (logo)
Universitair Medisch Centrum Utrecht (logo)
You are here: Home > Education > PhD > PhD theses > 2016 > January > Feenstra

Femke Feenstra

Tuesday 19 January 2016

Novel Bluetongue Vaccine Platform - NS3/NS3a knockout virus as disabled infectious single animal vaccine

Promotor: Prof. dr. R.J.M. Moormann
Defence: 19 January 2016

Bluetongue (BT) is a disease of wild and domestic ruminants, caused by the bluetongue virus (BTV). In principle, BTV is non-contagious and the main transmission route is by bites of certain species of Culicoides midges. The most observed symptoms of BTV infections are fever, oedema of the face and neck and oedema of the feet. The swollen cyanotic tongue, that gave the disease its name, is rare. These symptoms are mainly due to the haemorrhagic nature of the disease, with microvascular endothelial cells in several organs being the main target for virus replication. Mortality usually varies from 0 to 30%, but can be higher in certain breeds of sheep or wild dear species infected with highly virulent virus strains. The disease is often asymptomatic in cattle and in goats. Cattle are however important for transmission of BTV by a high and long-lasting viremia. Infection by BTV8, first detected in the Netherlands in 2006, is a remarkable exception, showing severe clinical signs in cattle.

BTV belongs to the family Reoviridae, genus Orbivirus, which means that is has a ten-segmented (Seg 1-10) double stranded RNA genome (~19 kb). The virus particle is non-enveloped, but architecturally complex, with a triple layered capsid shell of ~80 nm in diameter. Virions have an icosahedral shape with the outer capsid consisting of VP2 trimers, involved in cell entry, and VP5 trimers, involved in release from endosomes. The middle capsid layer consists of VP7 proteins, interacting with the VP3 inner capsid consisting. The replication complex consists of VP1 (polymerase), VP4 (capping enzyme) and VP6 (helicase), and likely forms a complex at the inner side of the VP3 layer. BTV also encodes at least four non-structural (NS) proteins, of which NS3 and its N-terminal truncated form NS3a (Seg-10) are viroporin-like membrane proteins involved in virus release mechanisms and also in the antagonism of the IFN-I response.

Historically, BT has been described as a disease of sheep in southern Africa. However, it now has a worldwide prevalence, with outbreaks in former BT free areas or with new serotypes reported regularly. The disease has been added to List A of the World Organization of Animal Health International Animal Health Code, due to the severity and fast spread of the disease, but also since symptoms can be similar to those of foot-and-mouth disease. The BTV serogroup consists of at least 27 different serotypes that show no or little cross protection. Multiple serotype situations exist in many countries, making BT control more difficult. In Europe, many outbreaks of BTV serotypes 1, 2 ,4, 9 and 16 have been reported in southern countries. In 2006, an incursion of BTV serotype 8 by a so far unidentified route has been reported in north-west Europe, expanding as far north as 53° N in Denmark. Currently, different BT serotypes are still circulating in Europe and recently an outbreak of BTV8 has been reported in France.

BT outbreaks induce large economic losses, which are not only caused by morbidity, mortality and production losses in affected animals, but are mainly due to trade restrictions in affected countries. The most effective method to diminish losses by BT is vaccination. Currently, live-attenuated and inactivated vaccines have been marketed. Although these vaccines have been shown to be effective in BT control, they also have their specific disadvantages. Live-attenuated vaccines can be incompletely attenuated, spread and reverse to virulence and inactivated vaccines have high costs and need booster vaccination. Further, differentiation of infected from vaccinated animals (DIVA) is not possible, which hampers monitoring and trade. These vaccines are thereby only available for a limited amount of serotypes. Therefore there is a need for next-generation types of BT vaccines. In this thesis such a novel vaccine candidate to control BT is described.

Previously, the expression of NS3/NS3a was shown to be not essential for BTV replication in vitro, by mutating both start codons using reverse genetics. NS3/NS3a knockout reduces virus growth and release in both mammalian and Culicoides cells, but this effect is stronger for Culicoides cells. In this thesis, we confirmed these findings, by making deletions in Seg-10, encoding NS3/NS3a. Indeed, BTV replicated without NS3/NS3a expression. However, the deleted RNA was replaced by several sequences originating from other viral segments. This happened each time when a deletion was introduced in the NS3/NS3a ORF, that aborted protein expression. We therefore concluded that RNA in the NS3/NS3a ORF is essential for virus replication. Further studies on the needs for Seg-10 in BTV replication, by changing NS3/NS3a codon usage, revealed that selection on Seg-10 RNA is stronger than on NS3/NS3a expression. Also, the functional RNA is at least partly conserved between orbiviruses, since exchange of Seg-10 RNA between orbiviruses did not result in RNA insertions. However, growth of BTV expressing NS3/NS3a from other orbiviruses shows that NS3/NS3a protein function is only partly conserved, mainly in mammalian cells. These findings, extended our knowledge on the needs for Seg-10 in BTV and led to the development of NS3/NS3a knockout virus as a BT vaccine candidate.

To examine the efficacy of NS3/NS3a knockout BTV, three different vaccine backbones were tested; the BTV1 labstrain, vaccine related BTV6 and virulent BTV8. All three backbones contained the same deletion in Seg-10, disabling NS3/NS3a expression and expressed VP2 originating from serotype 8. Groups of four sheep were vaccinated with these vaccine candidates and also with inactivated non-adjuvanted BTV8 as control for vaccine virus replication. Three weeks post vaccination, animals were challenged with virulent BTV8. All three vaccine candidates, including the one based on virulent BTV8, were avirulent and no viremia of vaccine virus was detected using sensitive RT-PCR. The BTV6 and BTV8 based vaccine candidates completely protected against challenge virus viremia and clinical signs, but the BTV1 based vaccine protected only partially. Comparison with the inactivated BTV8 vaccination, which was non-protective, revealed that the vaccine candidates replicated locally. These promising data led to a follow-up animal experiment using the BTV6 backbone. This time, animals were booster vaccinated with three weeks in between and were challenged with homologous BTV8 or heterologous BTV2 at nine weeks post infection. Again, vaccine virus did not induce clinical signs nor viremia, and protected against homologous challenge, but only partially against heterologous challenge. This does not only show vaccine efficacy longer after vaccination, but is also a good model to test serotype specific protection, not possible at three weeks post vaccination.
A competitive NS3 ELISA test was developed to check if differentiation of infected from vaccinated animals based on the presence or absence of NS3 antibodies is possible. NS3 antibodies were not detected after vaccination or after homologous challenge, showing the potential of the vaccine candidates for use in DIVA.

Up to this point, all tested vaccine candidates expressed VP2 of serotype 8. However, since there is hardly any cross protection for BTV, the vaccine needs to be applicable for all serotypes, starting with the European ones. NS3/NS3a knockout BTV6 could be serotyped with VP2 from serotype 1,2,4,8 and 9, but not with serotype 16. We therefore made BTV expressing chimeric VP2. The predicted VP2 ‘base’ originated from BTV1, but the outer part from BTV16. Vaccination of sheep with this chimeric virus raised antibodies against both BTV1 and BTV16, as shown by virus neutralization assays in vitro. Although no challenge was performed, these results suggest that chimeric VP2 can be a solution for problems with serotyping.

Since there is only local replication of vaccine virus in the vaccinated animal, vaccine spread by midges with subsequent chance on reassortment with field virus is highly unlikely. Therefore, our vaccine candidate has been named the disabled infectious, single animal (DISA) vaccine. However, vaccine spread is always a large concern for live-attenuated vaccines. We therefore wanted to reinforce the safety of our vaccine candidate, by studying the replication in Culicoides in vivo. In vitro we already saw that virus release in midge cells was almost absent and we therefore hypothesized that DISA replication in midges in vivo would be abolished. C.sonorensis midges were therefore injected with BTV with and without NS3/NS3a expression. Using RT-PCR, virus replication was only detected when NS3/NS3a was expressed. This strengthens the inability of DISA vaccine spread in the field and therefore the safety of this effective vaccine candidate.

These in vitro and in vivo data show that we now have a BT vaccine candidate that is safe regarding clinical signs and spread and that protects against clinical signs and viremia, enables DIVA and is applicable for multiple serotypes.