Challenges and Opportunities in Developing Oral Vaccines against Viral Diseases of Fish

Arun K. Dhar^1* and F. C. Thomas Allnutt²

¹Towson University, Biology Department, 8000 York Road, Towson, MD 2125

²Phycal Inc., 51 Alpha Park, Highland Heights, OH 44143

*Corresponding Author:

Arun K. Dhar
Towson University, Biology Department
8000 York Road, Towson, MD 2125
Tel: 858-395-7351
E-mail: adhar@towson.edu or arun_dhar@hotmail.com

Received date: November 04, 2011; Accepted date: November 22, 2011; Published date: November 25, 2011

Citation: Dhar AK, Allnutt FCT (2011) Challenges and Opportunities in Developing Oral Vaccines against Viral Diseases of Fish. J Marine Sci Res Development S1:003. doi: 10.4172/2155-9910.S1-003

Copyright: © 2011 Dhar AK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Visit for more related articles at Journal of Marine Science: Research & Development

Abstract

This review covers the current state of the art in oral vaccination of fish against viral diseases. It provides a comparison of the current state of fish vaccination, explores the various routes of vaccination, and then focuses on oral vaccination and the current state of this important and emerging vaccination strategy. A brief discussion is included of the essential research needed to support future widespread use of oral vaccination in fish aquaculture is also provided.

Keywords

Vaccination; Virus vaccine; Oral; Per os; Fish; Fish virus; Aquaculture

Introduction

Current status of global aquaculture

Worldwide consumption of fish is approximately 120 million metric tonnes. Of this overall production, aquacultured fish account for about 25% or 30 million metric tonnes [1]. Aquaculture’s contribution to fish production worldwide has doubled every decade since the 1980’s and this trend promises to continue indefinitely. This is partially a consequence of the pressure being placed on wild catch fisheries and the increasing world population. Wild catch fisheries are currently reaching their maximum output. Already 25% of the wildfish stocks are considered to be overfished or depleted, while an additional 52% are considered to be fully exploited [2]. This leaves little room for expansion of the wild catch to meet rising demand. As aquaculture is expected to supply more of the high quality protein needed for the earth’s rising population, the degree of intensification of industrial aquaculture management methods will put additional strains on the health of the animals in the system. As these intensive farming techniques dominate the industry, the potential for negative impacts on the environment will also increase. The high density culturing of fish in both freshwater and seawater aquaculture has already demonstrated that effective disease management is a key to profitability. Current vaccination methods are primarily limited to larger fish and focus on getting the fish to market using a single vaccination cycle. This approach is unacceptable, since it is really a race between disease and harvest that does little to reduce viral loads in the system. Ideally vaccines that provide protection for the entire life of the fish and are cost effective are the most desirable. These approaches will have to increasingly rely on earlier vaccination (while the fish are juveniles and small in size), booster vaccination to provide longer lasting protection, and rapid response vaccines to control the spread of disease and decrease negative effects of aquaculture on the environment (e.g., wild fish stocks).

Vaccinations are playing an increasingly important role in fish health management. The ideal vaccine must induce long lasting protection starting at an early age, prevent carrier formation, and be effective against a large number viral serotypes or viruses. Ideally, these vaccines will also allow the identification of a vaccinate from an infected (or previously infected) fish to aid in epidemiology and disease control. These attributes of a vaccine are most likely to be met either by a recombinant subunit vaccine or by an inactivated viral vaccine, as a live attenuated vaccine could potentially lead to carrier formation.While injectable vaccines dominate the market, alternative delivery routes to injection offer the tantalizing potential to deliver better results at reduced cost. However, these approaches also have additional challenges to overcome in order to provide effective disease control.

Control of a number of viral pathogens is essential for profitable and safe aquaculture (Table 1). The major viruses affecting aquaculture of salmonids are infectious salmon anemia (ISA), caused by Infectious Salmon Anemia Virus (ISAV), and infectious pancreatic necrosis (IPN), caused by Infectious Pancreatic Necrosis Virus (IPNV). ISAV is also important to a large number of other aquacultured species (Table 1) and is a continuing threat to salmon aquaculture [3]. Other critical viruses for salmonid aquaculture are the Salmon Alphaviruses (SAV) and Infectious Hematopoietic Necrosis Virus (IHNV). Commercially important salmonids other than salmon, such as rainbow trout, are also susceptible to these viruses. Spring Virema disease is caused by Spring Virema Carp Virus (SVCV) and affects a broad range of aquacultured species. Viral Nervous Necrosis Virus (VNNV) is important to sea bass and sea bream culture. Viral Hemorrhagic Septicemia Virus (VHSV) is an important pathogen of trout and turbot aquaculture.

Table 1: Major viral pathogens of farmed finfish.

An example of the importance of viral disease to aquaculture can be seen in the Chilean salmon industry. This industry underwent a rapid expansion, over the past decade but in 2010 was faced with an outbreak of ISA. This crisis took the production from 670,000 metric tonnes (2008) to about 100,000 metric tonnes in 2010 [4]. The outbreak was traced to production management methods and failure to vaccinate fish. Another recent example of the continuing impact of fish viruses on the industry is the report of ISAV in British Columbia sockeye salmon stocks where it has never before been observed [5].

The industry has responded to these challenges by a number of means. Management is a critical component to disease control and is invaluable to improved harvests and aquaculture generally. However, as open water aquaculture has become more intensive, the possibility of epidemic and pandemic outbreaks of viral disease has increased. Catastrophic outbreaks of ISA have devastated the salmon industry, such as the one in Cobscook and Passamaquoddy Bays, Maine, in 2001 resulting in depopulation of the entire area [6]. Such outbreaks have forced the industry to improve their biosecurity, management techniques and the supporting industry to develop other tools to directly address these diseases. A number of vaccines have been developed, as well as improved techniques for delivery of these vaccines at affordable prices. Commercially available vaccines are helping to improve the profit outlook of the industry (Table 2). The majority of vaccines are targeted at the salmonid industry with vaccines against SAV, IHNV, ISAV and IPNV. SVCV, while named for carp, is also important to catfish, wels, and rainbow trout, and has a vaccine based on inactivated virus marketed in Europe and delivered by injection. The majority of the available vaccines are based on inactivated virus and delivered by injection, primarily intraperitoneal. Recently introduced subunit vaccines are available targeting IPNV and ISAV (Table 2). The single DNA vaccine that is commercially available targets IHNV and is delivered by injection. The single oral vaccine targets ISA and is based on the hemagglutinin esterase protein of ISAV. However, these biotechnology based vaccines are being actively developed and it is anticipated that more will be introduced in the coming years as improved tools for disease control in aquaculture.

Table 2: Commercially available vaccines against viral diseases in farmed finfish.

Types of Vaccines

Inactivated viral vaccine

One type of vaccine is based on inactivated wildtype virus used as the antigen to induce an immune response. This was the earliest approach to fish vaccines and is still a reliable standard by which other vaccines are evaluated. The Alpha Jects^® Micro 1 ISA (Novartis) and Alpha Jects^® 1000 vaccines (Table 2) are examples of this type of vaccine, targeting ISA and IPN, respectively. Inactivated viral vaccines induce strong immune responses as they retain all of the virus’ surface exposed antigens as well as the inactivated genomic component that are important in induction of an immune response. Some fish viruses are not easily culturable, such as Lymphocystis Virus [7], preventing production of vaccines for these viruses based on whole inactivated virus. Alternative methods are necessary for vaccine production to lower the expense of production as well as to enable production of vaccines for unculturable or difficult to culture viruses.

Attenuated viral vaccine

The use of live viruses as vaccines has been used extensively in humans, such as the chicken pox vaccine. These are live virus vaccines that have been selected for cross reactivity (a less virulent virus that elicits an immune response to the target virus), or genetically modified and/or cultivated under conditions which disable viral virulence. As a result, the virus replicates in the target host albeit at a much lower rate compared to the wildtype virus and has no or reduced clinical signs. Attenuated viral vaccines typically provoke a strong and sustained immune response to the target disease. The current commercial vaccines are not based on attenuated viruses, but aquaculture vaccines have been reported that are based on attenuated viruses. An example is a vaccine attacking Koi Herpes Virus (KHV) based on attenuated Carp Interstitial Nephritis and Gill Necrosis Virus [8].

Subunit vaccine

Another type of vaccine is the subunit vaccine that emerged with the advent of molecular biology. Production of specific viral proteins in a recombinant system allowed the rapid production of focused vaccines based on a single viral antigen or small number of viral antigens. These vaccines allowed the high expression of the most highly antigenic proteins of the virus in bulk and delivery of highly purified preparations of these proteins as a vaccine. Initial work with subunit vaccines was not successful due to rapid degradation of the protein during processing, delivery or in the animals. However, rapid advances were made to stabilize the antigens and many subunit vaccines were developed (Table 2). Highly successful examples of subunit vaccines are the IPNV VP2 based vaccine from Microtek International and the ISAV recombinant hemagglutinin esterase gene from Centrovet (Table 2).

Some subunit vaccines were composed of the antigenic determinants of the virus but were not wholly effective in vaccine form. Different approaches were developed to improve the antigenic response of the animals. Formulation of the vaccines was the first focus and relied on improved stabilization of the proteins, addition of chemicals that induced the immune system generally or poised the immune system for better immune response, multiple antigens included in the vaccine (like the Trivalent subunit vaccine from Microtek; Table 2), and different formats to improve the display of the antigens and prevent their rapid degradation in the animal.

Subunit Vaccine – VLPs & SVPs: One of these formats is the viruslike particles (VLPs) or subviral particles (SVPs).The intrinsic ability of viral structural proteins to self-assemble into particles that mimic the native virus in both size and processing by the host have led to the development of a class of subunit vaccines based on VLPs [9,10,11]. VLPs have been expressed in bacteria, yeast, transgenic plants and cell culture. A number of human vaccines have been produced using this technology, such as the Gardasil vaccine by Merck. Recently, an effort has been made to extend this approach to production of vaccines for fish and shellfish [12,13]. The Shivappa study [13] targeted infectious pancreatic necrosis virus (IPNV) and used VLPs based on two of the capsid proteins, VP2 and VP3 [13]. These formed VLPs that were similar in size to the native virus and induced a strong immune response in salmon [13,14]. A more recent paper described production of an IPN vaccine based on the VP2 protein of IPNV [15]. In this study, a subunitbased particle was made that was smaller (22 nm) than the native virus (60 nm) but induced a strong anti-IPNV response in rainbow trout, this was referred to as a SVP. Another study demonstrated that a foreign epitope (epitope of a human oncogene c-myc) could be expressed on the subviral particle and induces an immune response to both IPNV and the foreign epitope [16]. This demonstrated that the IPNV SVP could be used as a platform to expressing heterologous epitope and to developing bi-/multivalent vaccines. Three other vaccines based on VP2 of IPNV are already marketed - IPNV (licensed in Chile, Centrovet, Chile), Norvax (Intervet-International BV, The Netherlands), SRS/ IPNV/Vibrio (licensed in Canada and Chile, Microtek International Inc., British Columbia, Canada).

Recombinant DNA vaccine

Recombinant DNA vaccines involve injecting an organism with histone-free (“naked”) DNA representing a certain gene from the pathogen itself. The expression of the protein representing the antigen is usually under the control of a strong promoter. The vaccine is administered by intramuscular injection or by gene gun bombardment of the epidermis. Upon delivery, the naked DNA is taken up by the muscle cells and the antigen protein is expressed. The antigen presenting cells, such as the dendritic cells at the site of injection, present the viral antigen via the MHC class I molecules to lymphocytes in the lymphnodes [17-19]. In mammals, it has been shown that DNA–based vaccines have some immunological advantage compared to traditional methods of vaccination because DNA-based vaccines induce strong and long-lasting humoral and cell-mediated immunity similar to those provided by live attenuated vaccine but without the risk of inadvertent infection [17]. DNA vaccines promise to provide a strong tool for aquaculture [20]. A DNA vaccine against infectious hematopoietic necrosis virus (IHNV) was the first effective DNA vaccine tested in fish (Table 2) [21]. Subsequently, DNA vaccines were tested against a number of fish viruses, including Infectious Hematopoietic Necrosis Virus [22,23], Viral Nervous Necrosis Virus (VNNV) [24], Infectious Pancreatic Necrosis Virus (IPNV) [25,26] and Spring Viremia Carp Virus (SVCV) [27,28].

Vaccination routes

Vaccination for aquatic species has three major routes of delivery: injection (intraperitoneal & intramuscular), immersion, and oral (per os). Most current vaccines are delivered by injection, predominantly intraperitoneal (Table 2). Each delivery route has its own advantages and limitations. These are discussed in the following sections.

Injection

The most common method for vaccination is via injection of the vaccine. This is usually done manually using a needle and intraperitoneal or intramuscular injection (Table 2). Alternative injection methods using devices such as compressed air have also been tested but are not in general use. This vaccination route requires fish of fairly large size and, even with automation, is stressful on the fish and labor intensive. These problems with the injection route have led to the development of a number of semi-automated methods where the fish are forced through a chute or enclosed space and, as they are wedged in are autoinjected with the vaccine. These are not in general use and tend to increase the amount of lesions at the site of injection.

Pros: Vaccination by injection provides a substantial number of positive attributes. This method delivers the most direct delivery of antigen to the immune system through intramuscular and intraperitoneal injection. The vaccine can be concentrated and delivered in the presence of adjuvants and other beneficial compounds (e.g., carriers, bacterial antigens/bacterial cells, and etc.) that could not be delivered by other methods. The antigens are protected in highly purified formats and can be easily stored in refrigerated form since they are so concentrated.

Cons: Injectable delivery also has a number of associated problems that make it desirable to develop alternative delivery methods for vaccines. The major issue with injectable vaccines is that they cannot be economically delivered multiple times demonstrated that. They are normally delivered at the beginning of grow out and the protection is intended to span the entire grow out cycle (until harvest).

The high cost of labor is a major concern for any injectable vaccine. Automated methods have been developed for injection such that the labor is decreased. However, injection causes damage at the site of injection such as tissue adhesion damage in interperitoneal injections [29,30]. The automated methods have an increase in this injection induced damage. In addition, often there are mortalities associated with the handling of fish during injection.

A major issue with injection vaccination is that it cannot be carried out early in the life of the fish since the immune system is not well developed in smaller fish. Additionally, juvenile and pre-smolt fish vaccination via injection route is not a feasible option due to the small size of these fish. As a result, there is a window for pathogen infection for unvaccinated fish from larval to just before grow out where increases in titer occur before these fish can be vaccinated by injection.

Delivery by immersion

A general and safe way to deliver a vaccine is by immersion of the fish in a solution containing the vaccine. The vaccine is delivered both to the skin and all mucosal surfaces accessible to antigen in the surrounding liquid. This is particularly effective in the induction of mucosal immunity but less so for humoral immunity [31]. Immersion as a route of vaccination is generally limited to smaller fish as the volumes of the immersion liquid or dip must be so large with very large fish. Improvements in the efficacy of immersion vaccination have been obtained through the use of ultrasound and the use of a hyperosmotic solution for the immersion medium.

Pros: Immersion vaccination is particularly suitable for smaller fish. This way they can be immunized in large batches and handling of small fish is kept to a minimum, and thus stress and mortalities are reduced. It is particularly suited to induction of a mucosal immune response [32,33]. It is an inexpensive and low stress method that requires little labor relative to injection as a method of vaccination. Since injection cannot be used for small fish, either oral or immersion delivery methods are more preferred routes for early vaccination.

Cons: This approach does not provide as strong an induction of humoral immune responses as injection vaccination. Fish must be collected and immersed in the antigen containing dip which can be time consuming and expensive with larger fish. It is not easy to deliver adjuvants and other immune stimulating compounds in an immersion system.

The vaccine is disbursed in a dilute solution and some of the vaccine may be lost during the delivery process (i.e., stay in the immersion fluid and be lost when the vaccination is over). The suspension in the aqueous medium also puts constraints on the type of material that may be in the vaccine such as solubility and stability.

Oral or Per Os Delivery

Pros: Oral delivery can be accomplished with any age fish, very low stress and is very inexpensive. Oral delivery does not require fish to be handled separately and therefore reduces the overall cost of the vaccination and any mortality associated with handling of the fish as seen in the case of injectable vaccine. With this reduction in labor and cost, oral vaccines can be used repetitively in the life of an aquacultured fish. Oral vaccines can be delivered to juvenile fish just developing humoral immunity, to fish prior to release in the grow out cages, and even during grow out in response to looming viral challenges. Oral vaccines can be delivered to fish at very low cost and without stress on the fish. Delivery of oral vaccines to fish which have already been immunized by immersion or injection as a booster vaccine can provide additional protection not achievable by a single vaccination [34,35]. Injection cannot be used for small fish, therefore either oral delivery or immersion are more preferred routes for early vaccination.

Cons: Induction of humoral immunity is not as strong as injection vaccination with current strategies. Antigen must be packaged and shelf stable for inclusion in feeds or treatments for rapid delivery of oral vaccines at any site where aquaculture is practiced.

Encapsulation of oral vaccines

A major challenge for successful commercialization of oral vaccines is how to preserve the antigen. This is a two-fold problem.

First, the antigen needs to be stable in the vaccine formulation that must, by definition, be put into a feed or feed supplement and delivered to the fish. This feed or feed supplement would most desirably be made in advance by the vaccine maker and be very stable on the shelf. Such an oral vaccine would be much bulkier than injectable vaccines, contain a mixture of materials that might destabilize the antigen (e.g., minerals, enzymes, and etc.), and would be less simple to store refrigerated (due mostly to mass).

Second, the antigen must be stable in the aquatic medium in which it is to be delivered and in the gut of the fish so that it is delivered in a form ready to induce a suitable immune response. The antigen would most preferably be poised or directed to the gut surface in order to facilitate this immune response.

Both of these issues have been addressed by encapsulation techniques to varying degrees. However, additional research is required in this area to improve oral vaccination. The recently introduced ISAV vaccine from Centrovet (Table 2) uses a proprietary microencapsulation system designed to protect the SVP-based ISAV hemagglutinin esterase antigen from destruction [34]. While the composition of this encapsulation matrix was not disclosed by these authors, other research has been on-going using materials such as alginate and chitosan to improve oral delivery of vaccines in fish [25,36,37], and work on this area continues.

The use of bioencapsulation for better delivery has been utilized for vaccines and other materials in aquaculture. An example is the use of Artemia to deliver vaccines in larval and juvenile stages that has been described for cultured koi, Cyprinus carpio [8]. The use of Artemia does not lend itself to scaled up delivery to larger fish, therefore alternative bioencapulation methods for delivery of subunit vaccines are required. These can be based on use of the expression host used for production of the antigen (e.g., recombinant bacteria or yeast). An example is the Centrovet ISAV vaccine where the HE antigen is expressed in yeast that are then microencapsulated in a proprietary formulation [34]. The use of the host used for production of the antigen to provide targeting of the antigen to the gut uptake sites is also a possibility. The host organism could be engineered to express targeting moieties on the cell surface that are recognized by the fish gut and presented to the immune system.

Current status and future direction

Due to the increasing scope of aquaculture (both in magnitude and types of fish under production), vaccines promise to become more important to the industry. Current commercial vaccines are predominantly injectables. However, there is a large amount of research underway to develop vaccines based on other routes of delivery, especially oral, that are less stressful, more flexible, and provide cost savings in the vaccine, labor and outcome. A brief description follows but, by necessity, will not cover all of the excellent research underway to fully enable these important tools for the aquaculture industry.

Experimental vaccines: Viral diseases of finfish are a major hurdle in finfish aquaculture and the periodic emergence of viral epizootics is a threat to sustainability of commercial finfish production. Among finfish, Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchusmykiss), sea bream (Dicentrachuslabrax), carp (Cyprinuscarpio), turbot (Scophthalmusmaximus), and catfish are the major species that are commercially cultivated worldwide. Diseases caused by viruses, such as infectious salmon anemia virus (ISAV), infectious pancreatic necrosis virus (IPNV), viral haemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV), viral nervous necrosis virus (VNNV) and salmonid alphavirus (SAV), are the major viral pathogens of finfish (Table 1). Disease outbreaks caused by these viruses have had major socio-economic and ecological impacts over the years and many of these viruses are now considered as notifiable pathogens by the Office International des Epizootics (OIE; http://www.oie.int). A recent identification of ISAV in wild salmon in British Columbia, Canada [5] shows the need and urgency for effective monitoring of this virus in wild population and developing strategies to contain its spread to commercial fisheries. Because viruses such as ISAV can cause high mortality, preventive methods such as vaccination are considered key measures to managing viral diseases in commercial fish farming. In recent years, a number of vaccines based on inactivated virus, recombinant subunit proteins, and DNA vaccines have been developed in the laboratories across the globe. Also a variety of delivery methods, such as intramuscular or intraperitoneal injection, immersion in water containing the vaccine or oral delivery via feed, have been tested. Although only a few of the vaccines tested in the laboratory have made it into the market place for commercial application so far (Table 2), there remains an enormous opportunity for bringing new vaccines into the market place and developing more efficacious vaccines. For example, an IPNV DNA vaccine expressing VP2 antigen and delivered orally provided almost 80% relative percent survival (RPS) upon challenge by an infectious homologous virus in rainbow trout fry (1-2 gm) and is very promising [25]. Currently there is no commercially available vaccine against diseases caused by VHSV and VNNV. However, a number of recent studies have reported candidate vaccines against these two diseases. For example, a DNA vaccine expressing the G-protein of VHSV delivered via intramuscular injection provided 100% RPS [38]. Similarly, VNNV vaccines based on inactivated virus, recombinant capsid protein, and a DNA vaccine expressing the viral capsid protein that are delivered either by intramuscular injection or via immersion have been reported [24,39-41]. An oral vaccine against ISAV is now commercially available [34] (Centrovet Ltd., Chile, Table 2), however its efficacy in preventing ISAV infection under field conditions is not yet publically available.

Oral vaccines: It is obvious from (Table 2) that oral vaccination has yet to contribute significantly to the overall vaccine pool available for aquaculture. The ISAV vaccine from Centrovet and the IPNV vaccine from Schering Plough-Intervet are the two oral vaccines commercially available. However, the potential for oral vaccines to contribute to fish health is tremendous. The simple delivery and low cost production methods associated with oral vaccines is a strong incentive to further develop oral vaccines in aquaculture. There are major hurdles to production of oral vaccines. Foremost of these hurdles being the stability of the immune stimulating agent in the gut and its successful induction of a strong humoral immune response by the orally delivered agent. Mucosal immunity is important and oral vaccines are strong inducers of mucosal immunity, however a complete vaccine should induce innate, mucosal and humoral immune responses.Oral vaccines should be amenable to all types of vaccines. That is subunit vaccines, attenuated virus based vaccines, nucleic acid vaccines and inactivated virus based vaccines.

One of the most promising delivery systems for subunit and nucleic acid based vaccines are recombinant organisms that do not require viral culturing and purification of antigens (or nucleic acids). A number of expression systems have been considered, but using yeast as a biofactory for vaccine production provides strong advantages over other systems: 1) The production of biologics in yeast is well understood in the industry and veterinary vaccine manufacturers are likely to subscribe to a system that does not require additional infrastructural modifications. 2) Since many yeast species and strains have GRAS status (Generally Regarded As Safe), the regulatory barrier for vaccines made in yeast tend to be lower than other manufacturing platforms such as bacteria or insect cell based system. 3) The cost of vaccine production in yeast-based manufacturing system is likely to be lower than associated with virus purification from yeast will be lower than that for insect cells. 4) The yeast expression system is a genetically well known and, thus, it is amenable to the genetic modification needed for high expression of the vaccine antigen protein. 5) In fish feed, whole yeast is used as an immunostimulant. Therefore, a vaccine made and delivered in whole yeast not only has general immune stimulatory benefit but also provides specific immunity against target pathogen(s).

Vaccination is already an important management tool for profitable aquaculture. Current methods predominately depend on injection for delivery. There is a need to develop alternative methods to reduce the cost of vaccination and provide a convenient means to vaccinate smaller fish as well as boost protection provided by injectable vaccination for stronger and longer lasting protection. The recent introduction of an oral vaccine against ISA and IPN for salmonids (Table 2) may signal a shift toward a more robust development of alternative vaccination strategies for aquaculture.

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