Background
AHS is a devastating disease of equids caused by an arbovirus of the Reoviridae family, genus Orbivirus. The virus is endemic to tropical and subtropical areas of Africa but it has repeatedly spread from its African habitat during the last century, causing severe outbreaks in Europe and most recently as far afield as Thailand, emphasizing that it is an active and ongoing global threat to the horse industry. Nine serotypes of AHS virus (AHSV) have been described, which poses a challenge for vaccine development. The virus is transmitted by haematophagous Culicoides midges. This increases the risk of an emergence of AHS in previously free regions driven by climate change, as shown for the closely related bluetongue virus (BTV) by its expansion from Africa to southern Europe since 2000 and the rapid spread in northwest Europe in 2006-2010. The infection of horses with AHSV leads to acute disease characterised by severe respiratory and circulatory impairment and is often fatal.
PPR constitutes one of the major hurdles to the improvement of small-ruminant production worldwide. It is extremely contagious and has a case fatality rate of up to 90%. Among domestic animals, goats and sheep are most affected, affecting the primary livelihood of 300 million rural families globally and causing an estimated US$ 2.1 billion in economic losses per year. The disease is endemic to most of Africa (except the extreme southern part of the continent), the Near East and in Asia extending as far as China. More recently, it has reached Turkey and Europe (Georgia and Bulgaria). This rapid expansion is now threatening more than 80% of the total sheep and goat population in the world, including in the EU. The causative agent is the PPR virus (PPRV, taxonomic name: Small ruminant morbillivirus), an enveloped RNA virus that is related to rinderpest, canine distemper and measles viruses. Following the lead of the successful rinderpest eradication campaign, the global eradication of PPR is targeted for 2030.
FMD is a severe, highly contagious viral disease of livestock that is the most important TAD on a global scale and severely disrupts animal production and trade in animals and their products. It affects cattle, swine, sheep, goats and other cloven-hoofed ruminants. FMD virus (FMDV) is believed to circulate in three quarters of the global livestock population primarily in Africa, the Middle East and Asia. Countries that are currently free of FMD without vaccination remain under constant threat of an incursion, but most of the costs attributed to FMD prevention and control are incurred bylow and lower-middle income countries. FMD is caused by an Aphthovirus of the family Picornaviridae. Seven serotypes of FMDV with many subtypes have been described. Recent outbreaks in Bulgaria and Israel illustrated the relevance of wild boar in FMD epidemiology and this will get worse as the populations of wild boar and feral pigs are expanding in the EU and around the globe.
Shortcomings of current vaccines
Traditional viral vaccines are produced by inactivation of virus cultures or gradual attenuation of virus by repeated passage in cell culture or laboratory animals. Inactivated vaccines are not without risk, as large amounts of virulent virus have to be grown prior to inactivation. Both inactivated and live-attenuated vaccines (LAVs) can stimulate an antibody response indistinguishable from the response to natural infection. This means that if vaccination is used in response to an outbreak, it is impossible to determine whether an animal has been infected despite prior vaccination, which prolongs the time it takes to declare the outbreak over and regain disease-free status. Hence, it can be more economically viable to cull infected and at-risk animals rather than vaccinate to break the chain of transmission, but control strategies that rely on culling alone are increasingly rejected by the public. The inability to distinguish between antibody responses to infection and vaccination also leads to disease-free countries imposing restrictions on the import of vaccinated animals from endemic regions.
In countries where AHSV is endemic, LAVs are widely used. Because these were developed empirically, the cause, extent and stability of the attenuation are not known and differ between strains. Incomplete attenuation and reversion to virulence in vaccinated animals can lead to clinical disease, uptake by midges and onward transmission of the virus. The segmented genome of AHSV means that reassortment with circulating field strains or between different vaccine strains can also result in the emergence of novel variants, as was reported from several countries in Africa. Importantly, these vaccines are not licensed for use outside of Africa. The current polyvalent live-attenuated AHS vaccine produced by Onderstepoort Biological Products in South Africa contains seven of the nine serotypes (5 and 9 are not included). These are split between two vials that, inconveniently, have to be administered on two separate occasions. Ideally, a single vaccine that provides protection against all serotypes of AHSV is required. Generally, polyvalent AHS vaccines are only required in endemic settings, because an outbreak in a previously free region will usually not involve more than one serotype. However, if a safe, efficacious, and affordable polyvalent vaccine was available, authorities in AHS-free regions may find it expedient to stockpile this product for use during the primary response to an outbreak pending the availability of a specific monovalent product. No licensed inactivated AHS vaccine is currently available, despite the fact that prototypes have been developed or are under development. Based on the experience with inactivated BTV vaccines in Europe, this approach was not chosen by the SPIDVAC consortium. Inactivated vaccines make it difficult to differentiate between infected and vaccinated animals, because they contain all proteins of the virus. Furthermore, to produce inactivated vaccines, the vaccine strains have to be selected carefully and adapted to growth in cell culture. This is laborious, time-consuming and requires a containment laboratory.
Similar to AHS, the control of PPR relies on classically attenuated LAVs. These are sufficiently effective that the Food and Agriculture Organization of the United Nations (FAO) and the OIE have developed a programme for its global eradication by 2030. When an outbreak of PPR has been contained in a free country, or when countries participating in the eradication programme stop vaccination and apply for official recognition of PPR-free status, the absence of PPRV circulation has to be demonstrated. Currently, this requires at least 24 months of intensive surveillance of susceptible animals because the available vaccines do not allow a serological differentiation between infection and vaccination. Innovative vaccines are required to make achieving official disease-free status quicker and more cost-effective and promote the eradication campaign.
In the past, FMD LAVs have been proposed that relied on eventual attenuation by repeated virus passage in cultured cells or small animals. The attenuated viruses created in this manner often unpredictably reverted to virulence and created rather than solved problems. Therefore, all FMD vaccines currently available in Europe are inactivated virus vaccines. Sophisticated and expensivepurification during manufacturing is required to remove or significantly reduce the amount of non-structural viral proteins in the final product. ELISA kits measuring the antibody response to these non-structural proteins, in particular 3ABC, can then be used to find evidence of infection in vaccinated animals. This method is, however, not reliable in endemic settings where animals are repeatedly vaccinated, and the purification technology and expertise are not readily available to all manufacturers of inactivated FMD vaccines. The wildlife reservoir of FMDV poses additional challenges. Vaccines that can be applied mucosally are the only option for immunization of wildlife, where neither classical inactivated vaccines nor particle or peptide vaccines can be used. The oral application of attenuated vaccines with bait blisters has been used successfully for wild boar (e.g., against classical swine fever).
The concept of DIVA
The DIVA (differentiation of infected from vaccinated animals) approach allows the detection of infection with wild-type viruses in vaccinated populations. The classical DIVA vaccine is missing an immunogenic component present in all natural variants of the respective pathogen that can be detected in a serological test. This is almost impossible with traditionally attenuated live virus vaccines or inactivated virus preparations and clearly, the currently available vaccines for AHS, PPR and FMD lack specific and sensitive DIVA features. A DIVA strategy is more feasible with subunit vaccines not containing the whole pathogen or purified virus particles not containing non-structural proteins. Similarly, vectored vaccines expressing only selected proteins of a pathogen can be designed. Biotechnological advances over the past few decades, including reverse genetics technology, have paved the way to recombinant viruses in which a specific immunogenic protein has been removed or replaced with a serologically distinguishable protein exhibiting the same viral function. An equally important part of the DIVA concept is the companion test to detect infected animals in vaccinated populations. Because of the high impact of false test results, these DIVA assays must be highly sensitive and highly specific.
For AHS, a broad range of vaccine candidates have been explored taking the DIVA concept into account from inactivated AHSV and protein subunit vaccines to viral vector vaccines and, more recently, to genetically modified AHSV, but none of these potential DIVA vaccines for AHS have reached the market. Similarly, many experimental vaccines against PPRV have been published, including some based on a LAV expressing a fluorescent protein generated by reverse genetics. While it can be attractive to develop such “positive marker” vaccines to confirm the vaccination status of animals, by itself this is not a DIVA vaccine. However, DIVA capacity can be combined with a positive marker by replacing a PPRV epitope associated with a strong immune response with a corresponding sequence from another morbillivirus that does not naturally infect ruminants. Not only does this limit the functional consequences of the genetic modification, but appropriate companion tests can then confirm vaccination by detection of antibodies to the foreign epitope that was added to the vaccine and infection by detecting antibodies to the PPRV epitope that was removed. For FMD, rather than laboriously removing non-structural proteins from the formulated vaccine, reverse genetics offers a more elegant and efficient path to a DIVA-capable vaccine. Similar to the approach described for PPR, highly immunogenic epitopes in the non-structural proteins can be modified or replaced with homologous sequences from other aphthoviruses to create a detectable difference between the antibody response to the vaccine and to the wild-type virus.