Development of a mucosal adjuvant for fish vaccination

Aquaculture is one of the fastest growing sectors that provide food to the expanding world population. Already ~50% of fish consumed by humans worldwide is produced in aquaculture, and this figure is projected to rise further. Like other food producing sectors, the sustainability of the aquaculture industry relies on good management of fish health and effective control of diseases. Outbreaks of infectious diseases can lead to heavy economic losses which may require many years to recover. During the last 30 years, vaccination has become an important means to improve fish resistance to bacteria, and has allowed a significant reduction in the use of antibiotics and toxic chemicals in fish farming, with tremendous benefits in terms of the environmental impact of the industry as well as improved fish health. The most effective way to achieve protection against diseases is to administer vaccines via intraperitoneal injection, partly due to the fact that antigens can be slowly released with the addition of oil based adjuvants for activation of the immune system. Despite the superior protection, this method causes stress to the fish, is labour intensive and can induce unwanted side-effects such as peritoneal adhesions, granulomas and even autoimmunity. However, immersion vaccination, where the fish are simply placed into a solution of diluted vaccine, has been shown to work in fish but to date is limited to a couple of bacterial pathogens and typically requires boosters for full protection, limiting its use. The need for effective adjuvants has largely driven injection vaccination, and this needs to be revisited with the appearance of several highly efficacious mucosal adjuvants being developed for use in mammals/humans. By linking a world leading mucosal adjuvant group at Oxford and a world leading fish immunology group at Aberdeen, this proposal plans to optimise the use of a novel mucosal adjuvant for use in fish, that has been developed by Professor Sattentau. Initially we will prepare stable formulations containing a killed bacterial pathogen (Yersinia ruckeri), that are suitable for testing in fish. We will use three formulations that vary in terms of the amount of adjuvant being used, and will compare the responses elicited in the fish (rainbow trout) with those elicited by a commercial immersion vaccine to this disease. We will examine the ability to enhance the expression of a number of important immune genes, thought to be involved in protective responses, as well as blood antibody levels. We will select the best formulation in terms of the responses elicited, to be used in a further experiment where the fish will be vaccinated and then exposed to the live pathogen, to see if the responses give good protection/immunity. If successful the results would have the potential to revolutionise immersion vaccination of fish. Enhanced immune responses/protection obtained with effective mucosal adjuvants may avoid the need for booster vaccination following immersion vaccination, or allow protective responses to be induced to diseases that currently require adjuvanted IP injection vaccination. We believe such an approach has the capacity for a step change in immersion (mucosal) vaccination technology for fish.

Mucosal/immersion delivery of vaccines to fish is possible but has been limited primarily due to the need for inclusion of adjuvants for effective protection to be elicited. This has driven the aquaculture industry towards (intraperitoneal) injection vaccines, to allow adjuvant inclusion. However, injection vaccines have several drawbacks; they stress the fish, are labour intensive and can induce unwanted side-effects and even autoimmunity. Recent research into mucosal adjuvants for mammals has shown promising results. For example, mice given a single intranasal administration of a nanoscale complex of polyethyleneimine (PEI) and viral glycoproteins have strong protection against viral infection. A major advantage of this type of formulation is that antigen and adjuvant can simply be mixed and will self-associate by reversible electrostatic interactions. In this proposal we will develop this approach for use in fish. Bacterial (Yersinia ruckeri)-PEI formulations will be developed that give polycationic nanoparticles after mixing, assessed by dynamic light scattering, scanning electron microscopy and zeta probe analysis. Antigen-adjuvant complexation will be assessed by isolation of nanoparticles and Western blotting, whilst formulation stability and dissociation will be measured by ELISA. Three formulations will be used for immersion vaccination of trout, and the immune responses elicited will be studied by transcript expression (by qPCR) of immune genes relevant to adaptive immunity during the first 10 days post-vaccination, and antibody levels (by ELISA) at 6, 8 and 10 weeks post-vaccination (antibody responses take weeks to develop in fish). The best formulation will then be used in a vaccine-challenge experiment where immune responses will again be examined, post-challenge, by qPCR and immunohistochemistry (using MoAbs to IgM/T and IFN-gamma/IL-22, the latter key cytokines important in Th1 and Th17 responses respectively), and disease resistance determined.

The development of novel vaccination strategies for salmonid fish will have long lasting economic and societal benefits by increasing the sustainability and growth of the UK fish farming industry. Beneficiaries will include aquaculture, government, private enterprise and, ultimately, the wider public as consumers. Aquaculture currently provides nearly half of all fish consumed globally and is one of the fastest growing animal-food producing industries. Scotland is the third largest producer of Atlantic salmon and in 2013 production reached 163.2 tons, making it the largest food export for Scotland with an annual retail value of > £1 billion. With the human population increasing and with wild fish stocks harvested to capacity (or decreasing), aquaculture will be placed under considerable pressure to meet consumer demands. As fish farming continues to expand and intensify, the frequency and severity of disease outbreaks have the potential to increase if effective control measures such as vaccination are not in place.
Although emphasis has been placed on prophylaxis, primarily vaccines, to successfully prevent major epizootics, the current vaccines for salmon are not ideal as most are administered via intraperitoneal (IP) injection due to the need for inclusion of adjuvants. Injection vaccination is costly and labour intensive and can cause collateral losses either by favouring infection with opportunistic pathogens (e.g. Saprolegnia) or from side effects of the vaccine itself (e.g. adhesions). In addition, the adjuvanted vaccines pose a major occupational health risk to the vaccinators, with granulomas developing at the sites of accidental needle pricks. Developing alternative vaccine delivery systems would address many of the problems associated with IP injection with the potential to 1) decrease the costs of fish vaccination, 2) improve vaccinator safety, 3) reduce losses associated with side effects and/or opportunistic infections, and as a result 4) improve animal welfare. The Scottish governments National Marine Plan proposes a 50% increase in aquaculture production by 2020, and this will require significant technological innovation to ensure sustainability and prevent disease. Initiatives supported by the UK government to discover and apply new technologies to improve animal welfare (i.e. NC3Rs) will profit from this research. Through our close links to global (fish) vaccine companies (eg Zoetis, MSD, Lilly/Novartis), our findings will have a direct route to commercialisation. Given that the technology could be transferable to other fish species, it has broad long-term commercial value if successful. Thus, ultimately this project aims to help maintain the UK as a world leader in aquaculture.